RESUMO
Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.
Assuntos
Anoctamina-1 , Bestrofinas , Gânglios Espinais , Hiperalgesia , Privação do Sono , Medula Espinal , Animais , Feminino , Masculino , Ratos , Anoctamina-1/metabolismo , Bestrofinas/metabolismo , Canais de Cálcio Tipo L , Canais de Cloreto/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/genética , Hiperalgesia/metabolismo , Ratos Wistar , Privação do Sono/metabolismo , Privação do Sono/complicações , Sono REM/fisiologia , Medula Espinal/metabolismoRESUMO
Bestrophin-1, a calcium-activated chloride channel (CaCC), is involved in neuropathic pain; however, it is unclear whether it has a dimorphic role in female and male neuropathic rats. This study investigated if 17ß-estradiol and estrogen receptor alpha (ERα) activation regulate bestrophin-1 activity and expression in neuropathic rats. Neuropathic pain was induced by L5-spinal nerve transection (SNT). Intrathecal administration of CaCCinh-A01 (.1-1 µg), a CaCC blocker, reversed tactile allodynia induced by SNT in female but not male rats. In contrast, T16Ainh-A01, a selective anoctamin-1 blocker, had an equal antiallodynic effect in both sexes. SNT increased bestrophin-1 protein expression in injured L5 dorsal root ganglia (DRG) in female rats but decreased bestrophin-1 protein in L5 DRG in male rats. Ovariectomy prevented the antiallodynic effect of CaCCinh-A01, but 17ß-estradiol replacement restored it. The effect of CaCCinh-A01 was prevented by intrathecal administration of MPP, a selective ERα antagonist, in rats with and without prior hormonal manipulation. In female rats with neuropathy, ovariectomy prevented the increase in bestrophin-1 and ERα protein expression, while 17ß-estradiol replacement allowed for an increase in both proteins in L5 DRG. Furthermore, ERα antagonism (with MPP) prevented the increase in bestrophin-1 and ERα protein expression. Finally, ERα activation with PPT, an ERα selective activator, induced the antiallodynic effect of CaCCinh-A01 in neuropathic male rats and prevented the reduction in bestrophin-1 protein expression in L5 DRG. In summary, data suggest ERα activation is necessary for bestrophin-1's pronociceptive action to maintain neuropathic pain in female rats. PERSPECTIVE: The mechanisms involved in neuropathic pain differ between male and female animals. Our data suggest that ERα is necessary for expression and function of bestrophin-1 in neuropathic female but not male rats. Data support the idea that a therapeutic approach to relieving neuropathic pain must be based on patient's gender.
Assuntos
Bestrofinas , Estradiol , Receptor alfa de Estrogênio , Gânglios Espinais , Neuralgia , Caracteres Sexuais , Animais , Masculino , Feminino , Neuralgia/metabolismo , Neuralgia/tratamento farmacológico , Ratos , Receptor alfa de Estrogênio/metabolismo , Estradiol/farmacologia , Estradiol/administração & dosagem , Bestrofinas/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Ratos Sprague-Dawley , Hiperalgesia/metabolismo , Hiperalgesia/tratamento farmacológico , Modelos Animais de Doenças , OvariectomiaRESUMO
Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PERSPECTIVE: SNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.
Assuntos
Hiperalgesia , Neuralgia , Ratos , Animais , Bestrofinas/metabolismo , Hiperalgesia/metabolismo , Ratos Sprague-Dawley , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Neuralgia/metabolismo , Nervos Espinhais/lesões , Ligadura , Canais de Cloreto/metabolismo , Gânglios Espinais/metabolismoRESUMO
Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.
Assuntos
Neurônios Aferentes , Ácido gama-Aminobutírico , Axônios , Gânglios Espinais , Neuroglia , Receptores de GABA-ARESUMO
In this study we determined the role of Ca(2+)-activated chloride channels (CaCC) in acute and chronic nociceptive responses elicited by 1% formalin. Formalin injection produced a typical pattern of flinching behavior for about 1h. Moreover, it produced secondary allodynia and hyperalgesia in the ipsilateral and contralateral paws for at least 6 days. Local peripheral and intrathecal pre-treatment (-10 min) with the non-selective and selective CaCC blockers niflumic acid and CaCCinh-A01, respectively, prevented formalin-induced flinching behavior mainly during phase 2 of the formalin test. Furthermore, niflumic acid and CaCCinh-A01 also prevented in a dose-dependent manner the long-lasting evoked secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Moreover, local peripheral and intrathecal post-treatment (on day 6) with both CaCC blockers decreased the established formalin-induced secondary mechanical allodynia and hyperalgesia behavior in both paws. CaCC anoctamin-1 and bestrophin-1 were detected in the dorsal root ganglia. Formalin injection increased anoctamin-1, but not bestrophin-1 protein levels at 6 days. Intrathecal injection of the CaCC inhibitor CaCCinh-A01 prevented formalin-induced anoctamin-1 increase. Data suggest that peripheral and spinal CaCC, and particularly anoctamin-1, participates in the acute nociception induced by formalin as well as in the development and maintenance of secondary mechanical allodynia and hyperalgesia. Thus, CaCC activity contributes to neuronal excitability in the process of nociception induced by formalin.