RESUMO
Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.
Assuntos
Dor/fisiopatologia , Receptores Opioides delta/fisiologia , Receptores Opioides mu/fisiologia , Analgesia , Analgésicos Opioides/farmacologia , Animais , Técnicas de Introdução de Genes , Temperatura Alta , Masculino , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Nociceptores/fisiologia , Dor/induzido quimicamente , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Medula Espinal/patologia , Medula Espinal/fisiologia , Substância P/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
BACKGROUND: Prolonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways. RESULTS: Using in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode. CONCLUSIONS: Our findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.
Assuntos
Nociceptores/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/citologia , Canais de Cátion TRPC/genética , Tapsigargina/farmacologia , Fatores de Tempo , Fosfolipases Tipo C/metabolismoRESUMO
Evidence suggesting the involvement of P2X2 and P2X3 in chronic pain has been obtained mostly from rodent models. Here we show that rodents may be poor predictors of P2X3 pharmacology in human. We demonstrate that monkey and human dorsal root ganglion (DRG) neurons do not express appreciable levels of P2X2 subunit, contrary to rat sensory neurons. Additionally, we report functional P2X3 activity in monkey DRG neurons and confirm the absence of functional P2X2/3 receptors. Interestingly, native P2X3 receptors in rat and monkey DRGs show similar agonist potency, but different antagonist potencies for TNP-ATP [2-O-(2,4,6-trinitrophenyl)-ATP] and RO51. This unexpected difference in antagonist potency was confirmed by comparing rat and human P2X3 receptors in HEK293 cells. Mutagenesis studies reveal that two extracellular residues, A197 and T202, are synergistically responsible for the potency drop in primate P2X3 receptors. These results uncover species-specific P2X3 pharmacology and identify key mechanisms impacting the translatability of potential analgesics targeting P2X3 receptors.
Assuntos
Expressão Gênica/fisiologia , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adulto , Análise de Variância , Animais , Contagem de Células , Células Cultivadas , Criança , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Gânglios Espinais/citologia , Expressão Gênica/efeitos dos fármacos , Humanos , Macaca fascicularis , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Mutagênese/genética , Técnicas de Patch-Clamp , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X/genética , Especificidade da Espécie , Transfecção , Adulto JovemRESUMO
The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.
Assuntos
Arteríolas/metabolismo , Química Encefálica/genética , Regulação da Expressão Gênica , Genes Reporter , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPV/biossíntese , Animais , Arteríolas/química , Humanos , Hipotálamo/química , Hipotálamo/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Miócitos de Músculo Liso/química , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/fisiologia , Vasoconstrição/genética , Vasodilatação/genéticaRESUMO
BACKGROUND: Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions. RESULTS: Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,ß-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,ß-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,ß-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,ß-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude. CONCLUSION: Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C/metabolismo , Receptores Purinérgicos P2X/metabolismo , Canais de Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Hibridização In Situ , Masculino , Canal de Sódio Disparado por Voltagem NAV1.3 , Naftalenos/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Estaurosporina/farmacologiaRESUMO
Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.
Assuntos
Encefalinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Cálcio/metabolismo , Embrião de Mamíferos/fisiologia , Encefalinas/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Hibridização In Situ , Ligantes , Dados de Sequência Molecular , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Peptídeos Opioides/metabolismo , Filogenia , Ligação Proteica , Precursores de Proteínas/genética , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Alinhamento de Sequência , Distribuição TecidualRESUMO
The present study demonstrates that perikaryaldelta-opioid receptors (deltaORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal deltaORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog omega-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of mu-opioid receptor (muOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of deltaORs observed after sustained morphine is attributable to stimulation of muORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Adelta fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 microm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal deltaORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to muOR agonists.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Morfina/farmacologia , Medição da Dor/efeitos dos fármacos , Receptores Opioides delta/análise , Receptores Opioides delta/biossíntese , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Masculino , Morfina/uso terapêutico , Dor/tratamento farmacológico , Dor/metabolismo , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/fisiologiaRESUMO
It is generally accepted that the voltage-gated, tetrodotoxin-sensitive sodium channel, Na(V)1.7, is selectively expressed in peripheral ganglia. However, global deletion in mice of Na(V)1.7 leads to death shortly after birth (Nassar et al. [2004] Proc. Natl. Acad. Sci. U. S. A. 101:12706-12711), suggesting that this ion channel might be more widely expressed. To understand better the potential physiological function of this ion channel, we examined Na(V)1.7 expression in the rat by in situ hybridization and immunohistochemistry. As expected, highest mRNA expression levels are found in peripheral ganglia, and the protein is expressed within these ganglion cells and on the projections of these neurons in the central nervous system. Importantly, we found that Na(V)1.7 is present in discrete rat brain regions, and the unique distribution pattern implies a central involvement in endocrine and autonomic systems as well as analgesia. In addition, Na(V)1.7 expression was detected in the pituitary and adrenal glands. These results indicate that Na(V)1.7 is not only involved in the processing of sensory information but also participates in the regulation of autonomic and endocrine systems; more specifically, it could be implicated in such vital functions as fluid homeostasis and cardiovascular control.
Assuntos
Sistema Nervoso Autônomo/metabolismo , Sistema Endócrino/metabolismo , Canais de Sódio/metabolismo , Animais , Linhagem Celular Transformada , Sistema Nervoso Central/metabolismo , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.7 , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/genéticaRESUMO
The expression of the galanin receptor-1 and -2 (Gal(1) and Gal(2)) messenger ribonucleic acids (mRNAs) was studied in the lower spinal cord of rat by means of in situ hybridization, using ribonucleic acid probes (riboprobes). Naïve rats as well as rats with unilateral axotomy of the sciatic nerve or unilateral inflammation of the hindpaw were analyzed. In naïve rats, numerous Gal(1) mRNA-positive (+) neurons were detected in lamina (L) I-III. In addition, several Gal(1) mRNA(+) neurons were seen in deeper layers, including the ventral horns, area X, and the lateral spinal nucleus. In contrast, few and comparatively weakly labeled Gal(2) mRNA(+) neurons were observed, mostly in the ventral horns and in area X, with fewer in the dorsal horn and in the sympathetic and parasympathetic intermediate lateral cell columns. Axotomy induced a strong increase in intensity and number of Gal(2) mRNA(+) motoneurons ipsilateral to the lesion. In contrast, nerve cut or hindpaw inflammation did not alter the expression of Gal(1) or Gal(2) in the dorsal horn. The present (and previous) results suggest that galanin, acting through Gal(1) and Gal(2) receptors, has a modulatory role on spinal excitability, not only via interneurons in superficial dorsal horn, but also on neurons in deep layers and area X, as well as on the sympathetic and parasympathetic outflow. Furthermore, the nerve injury-induced Gal(2) upregulation in motor neurons suggests a role for galanin in survival/regeneration mechanisms.
Assuntos
Regulação da Expressão Gênica/fisiologia , Expressão Gênica/fisiologia , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/metabolismo , Medula Espinal/metabolismo , Animais , Axotomia/métodos , Hibridização In Situ/métodos , Inflamação/metabolismo , Região Lombossacral , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Galanina/genética , Receptor Tipo 2 de Galanina/genética , Medula Espinal/citologiaRESUMO
An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.
Assuntos
Morfina/farmacologia , Entorpecentes/farmacologia , Receptores Opioides delta/metabolismo , Medula Espinal/metabolismo , Animais , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hibridização In Situ , Região Lombossacral , Masculino , Microscopia de Fluorescência , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Células do Corno Posterior/metabolismo , Transporte Proteico , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/agonistas , Receptores Opioides delta/biossíntese , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Rizotomia , Medula Espinal/anatomia & histologia , Regulação para CimaRESUMO
To assess the validity of rodent models for investigating the role of delta opioid receptors (DOR) in analgesia, the distribution of DOR binding and mRNA were compared between rodent and primate spinal cord and dorsal root ganglia (DRG), using receptor autoradiography and in situ hybridization, respectively. In mouse and rat spinal cord, [(125)I]-deltorphin-labeled DOR binding sites were detected throughout the gray matter. In contrast, in primate and particularly in human spinal cord, DOR binding was mainly present in laminae I-II, with little to no binding in deeper layers. Accordingly, in rodent spinal cord, DOR mRNA was expressed by a large number of neurons distributed throughout the ventral and dorsal horns, whereas in the primate, DOR expression was significantly lower, as evidenced by a moderate number of labeled cells throughout the gray matter in monkey and by only few labeled cells in human, mainly in Clarke's column and lamina IX. Major species differences in DOR expression were also observed in primary afferent cells bodies. In rat DRG, intense DOR mRNA hybridization was primarily observed over large ganglion cells immunopositive for neurofilament 200. In contrast, in monkey and human DRG, DOR mRNA was primarily detected over small and medium-sized ganglion cells. These results demonstrate major differences in the expression and distribution of DOR in the spinal cord and DRG between mammalian species. Specifically, they point to a progressive specialization of DOR toward the regulation of primary somatosensory, namely nociceptive, inputs during phylogeny and suggest that the effects of DOR agonists in rodents may not be entirely predictive of their action in humans.
Assuntos
Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/fisiologia , Filogenia , Receptores Opioides delta/biossíntese , Medula Espinal/metabolismo , Animais , Sítios de Ligação/fisiologia , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/genética , Especificidade da EspécieRESUMO
Recent molecular cloning studies have established the existence of a third rat galanin receptor subtype, GalR3, however its precise distribution in the mammalian central nervous system (CNS) is not well established. In the present study, we examined the regional and cellular distribution of GalR3 mRNA in the CNS of the rat by in situ hybridization. Our findings indicate that GALR3 mRNA expression in the rat brain is discrete and highly restricted, concentrated mainly in the preoptic/hypothalamic area. Within the hypothalamus, GalR3 expression was confined to the paraventricular, ventromedial and dorsomedial hypothalamic nuclei. In addition to these hypothalamic nuclei, GalR3 mRNA-expressing cells were observed in the medial septum/diagonal band of Broca complex, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus, the periaqueductal gray, the lateral parabrachial nucleus, the dorsal raphe nucleus, the locus coeruleus, the medial medullary reticular formation and in one of the circumventricular organs, the subfornical organ. In the spinal cord, a faint but specific ISH signal was observed over the laminae I-II with a few moderately labeled cells distributed in laminae V and X. The neuroanatomical distribution of GalR3 suggests it might be involved in mediating documented effects of galanin on food intake, fluid homeostasis, cardiovascular function and nociception.
Assuntos
Encéfalo/metabolismo , Receptores de Neuropeptídeos/metabolismo , Medula Espinal/metabolismo , Animais , Autorradiografia , Encéfalo/anatomia & histologia , Hibridização In Situ , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Galanina , Receptores de Neuropeptídeos/genética , Medula Espinal/anatomia & histologiaRESUMO
Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.
Assuntos
Mecanorreceptores/fisiologia , Neurônios/fisiologia , Nociceptores/fisiologia , Receptores Opioides delta/metabolismo , Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Canais de Cálcio/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Mecanorreceptores/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Dor/fisiopatologia , Medula Espinal/efeitos dos fármacosRESUMO
Comparison of human, rat and mouse cannabinoid CB(2) receptor primary sequences has shown significant divergence at the mRNA and protein sequence level, raising the possibility of species specific pharmacological properties. Additionally, given the importance of the dog as a non-rodent species for predicting human safety during the drug development process, we cloned the dog CB(2) receptor gene and characterized its in-vitro pharmacological properties in a recombinant expression system. A 1.1 kb dog peripheral cannabinoid receptor (dCB(2)) fragment encoding a 360 amino acid protein was cloned from dog spleen cDNA. Analysis of the cloned dCB(2) polypeptide sequence revealed that it shares between 76 and 82% homology with rat, mouse, human and predicted chimpanzee cannabinoid CB(2) receptors. The dog CB(2) receptor expressed in CHO cells displayed similar binding affinities for various synthetic and endogenous cannabinoids as compared to those measured for the human and rat cannabinoid CB(2) receptors. However, these ligands exhibited altered functional potencies and efficacies for the dog cannabinoid CB(2) receptor, which was also found to be negatively coupled to adenylate cyclase activity. These complex pharmacological differences observed across species for the cannabinoid CB(2) receptor suggest that caution should be exerted when analyzing the outcome of animal efficacy and safety studies, notably those involving cannabinoid CB(2) receptor targeting molecules tested in the dog.
Assuntos
Cães/genética , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Sequência de Aminoácidos , Animais , Ácidos Araquidônicos/farmacologia , Sequência de Bases , Benzoxazinas/farmacologia , Ligação Competitiva , Células CHO , Canfanos/farmacologia , Clonagem Molecular , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Cicloexanóis/farmacologia , Dronabinol/análogos & derivados , Dronabinol/farmacologia , Endocanabinoides , Humanos , Camundongos , Dados de Sequência Molecular , Morfolinas/farmacologia , Naftalenos/farmacologia , Pan troglodytes , Alcamidas Poli-Insaturadas/farmacologia , Pirazóis/farmacologia , Ratos , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Alinhamento de SequênciaRESUMO
Sensory neuron-specific receptors (SNSRs) belong to a large family of GPCRs, known as Mrgs (Mas-related genes), many of which are preferentially expressed in primary afferent nociceptors. Selective SNSR agonists produce pain-like behaviors in rats, showing that SNSR activation is sufficient to produce pain. However, it is unknown whether SNSR activation is necessary for pain either in the normal condition or in pathological pain states. Here we used small interfering RNA (siRNA) to acutely knockdown rat SNSR1 and test the hypothesis that this receptor mediates pain responses. Administration of siRNA to the lumbar spinal cord in rats dose-dependently knocked down rSNSR1 mRNA and protein and abolished heat hyperalgesia evoked by intradermal administration of specific rSNSR1 agonists. In rats with levels of rSNSR1 knockdown sufficient to block responses to the SNSR1 agonists, there was no effect on normal pain responses, but there was a significant reduction of heat hyperalgesia in an inflammatory pain model (Complete Freund's Adjuvant), supporting a role for rSNSR1 in inflammatory pain. Further in vivo studies revealed that SNSR1 knockdown had no effect on responses to intradermal capsaicin, a selective TRPV1 agonist. In contrast, a selective TRPV1 antagonist abolished heat hyperalgesia produced by an SNSR agonist, suggesting that TRPV1 receptors mediate rSNSR1-evoked responses. We also found that rSNSR1-like immunoreactivity, like TRPV1, is localized in the superficial dorsal horn of the spinal cord. We propose that rSNSR1 represents a new member of the receptors expressed on chemosensitive nociceptors responsible for detecting the "inflammatory soup" of mediators generated by tissue damage.
Assuntos
Vias Aferentes/metabolismo , Hiperalgesia/metabolismo , Inflamação/metabolismo , Neuralgia/metabolismo , Neurônios Aferentes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPV/metabolismo , Tato , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.
Assuntos
Códon de Terminação/genética , Mutação , Insensibilidade Congênita à Dor/genética , Canais de Sódio/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.7 , Dor/genética , Dor/fisiopatologia , Insensibilidade Congênita à Dor/fisiopatologia , Linhagem , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/metabolismoRESUMO
We examined the effect of galanin (10(-15) - 10(-7) M) on dispersed, mainly small-sized dorsal root ganglion (DRG) neurons in adult rats using whole-cell patch-clamp. Galanin and AR-M1896, a selective galanin type 2 receptor (GalR2) agonist, both significantly increased the number of action potentials in response to current pulses in 77% of the neurons, indicating an increase in excitability. Galanin also caused a rise in input resistance, decreased the holding current for -60 mV and depolarized the resting potential. In addition, Ca(2+) currents elicited by voltage steps were significantly increased by both galanin and AR-M1896 in nearly 70% of the cells. This enhancement was observed in 30% of the neurons in the presence of nimodipine or omega-conotoxin, but in each case approximately 60% less than without blocking either N- or L-type Ca(2+) channels, indicating modulation of both types of Ca(2+) channels. The percentage of small- and medium-sized neurons expressing GalR2 mRNA in DRGs in situ was similar to that showing increased excitability and Ca(2+) current after galanin application, i.e. approximately 70-80% of the neurons. The findings suggest that GalR2 has a role in controlling both the excitability, probably by inhibition of GIRK or leak K(+) channels, and Ca(2+) entry in a large population of presumably nociceptive neurons. The combination of the two effects, which possibly arise from separate biochemical pathways, would increase excitability and enhance intracellular Ca(2+) signalling which would enhance sensory transmission. These mechanisms involving GalR2 receptors may underlie the pronociceptive effects of galanin described in the literature.
Assuntos
Cálcio/metabolismo , Galanina/farmacologia , Gânglios Espinais/citologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Impedância Elétrica , Galanina/agonistas , Hibridização In Situ , Masculino , Neurônios/classificação , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/genética , Receptor Tipo 2 de Galanina/metabolismo , Isótopos de Enxofre/metabolismoRESUMO
Cannabinoids have been considered for some time as potent therapeutic agents in chronic pain management. Central and systemic administration of natural, synthetic and endogenous cannabinoids produce antinociceptive and antihyperalgesic effects in both acute and chronic animal pain models. Although much of the existing data suggest that the analgesic effects of cannabinoids are mediated via neuronal CB1 receptors, there is increasing evidence to support a role for peripheral CB2 receptors, which are expressed preferentially on immune cells. As yet, little is known about the central contribution of CB2 in neuropathic pain states. We report here that chronic pain models associated with peripheral nerve injury, but not peripheral inflammation, induce CB2 receptor expression in a highly restricted and specific manner within the lumbar spinal cord. Moreover, the appearance of CB2 expression coincides with the appearance of activated microglia.