RESUMO
Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.
RESUMO
BACKGROUND AND PURPOSE: Neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) is a novel hypnotic and voltage-dependent blocker of T-type calcium channels. Here, we examine its potential analgesic effects and adjuvant anaesthetic properties using a post-surgical pain model in rodents. EXPERIMENTAL APPROACH: Analgesic properties of 3ß-OH were investigated in thermal and mechanical nociceptive tests in sham or surgically incised rats and mice, with drug injected either systemically (intraperitoneal) or locally via intrathecal or intraplantar routes. Hypnotic properties of 3ß-OH and its use as an adjuvant anaesthetic in combination with isoflurane were investigated using behavioural experiments and in vivo EEG recordings in adolescent rats. KEY RESULTS: A combination of 1% isoflurane with 3ß-OH (60 mg·kg-1 , i.p.) induced suppression of cortical EEG and stronger thermal and mechanical anti-hyperalgesia during 3 days post-surgery, when compared to isoflurane alone and isoflurane with morphine. 3ß-OH exerted prominent enantioselective thermal and mechanical antinociception in healthy rats and reduced T-channel-dependent excitability of primary sensory neurons. Intrathecal injection of 3ß-OH alleviated mechanical hyperalgesia, while repeated intraplantar application alleviated both thermal and mechanical hyperalgesia in the rats after incision. Using mouse genetics, we found that CaV 3.2 T-calcium channels are important for anti-hyperalgesic effect of 3ß-OH and are contributing to its hypnotic effect. CONCLUSION AND IMPLICATIONS: Our study identifies 3ß-OH as a novel analgesic for surgical procedures. 3ß-OH can be used to reduce T-channel-dependent excitability of peripheral sensory neurons as an adjuvant for induction and maintenance of general anaesthesia while improving analgesia and lowering the amount of volatile anaesthetic needed for surgery.
Assuntos
Analgesia , Canais de Cálcio Tipo T , Neuroesteroides , Animais , Hiperalgesia/tratamento farmacológico , Hipnóticos e Sedativos , Camundongos , Dor Pós-Operatória/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , RoedoresRESUMO
It has been established that Ca(V)3.2 T-type voltage-gated calcium channels (T-channels) play a key role in the sensitized (hyperexcitable) state of nociceptive sensory neurons (nociceptors) in response to hyperglycemia associated with diabetes, which in turn can be a basis for painful symptoms of peripheral diabetic neuropathy (PDN). Unfortunately, current treatment for painful PDN has been limited by nonspecific systemic drugs with significant side effects or potential for abuse. We studied in vitro and in vivo mechanisms of plasticity of Ca(V)3.2 T-channel in a leptin-deficient (ob/ob) mouse model of PDN. We demonstrate that posttranslational glycosylation of specific extracellular asparagine residues in Ca(V)3.2 channels accelerates current kinetics, increases current density, and augments channel membrane expression. Importantly, deglycosylation treatment with neuraminidase inhibits native T-currents in nociceptors and in so doing completely and selectively reverses hyperalgesia in diabetic ob/ob mice without altering baseline pain responses in healthy mice. Our study describes a new mechanism for the regulation of Ca(V)3.2 activity and suggests that modulating the glycosylation state of T-channels in nociceptors may provide a way to suppress peripheral sensitization. Understanding the details of this regulatory pathway could facilitate the development of novel specific therapies for the treatment of painful PDN.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Neuropatias Diabéticas/tratamento farmacológico , Glicosilação/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Animais , Asparagina/metabolismo , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Células HEK293 , Humanos , Camundongos , Camundongos Obesos , Neuraminidase/metabolismo , Nociceptores/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Proteínas Recombinantes , TransfecçãoRESUMO
Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Biofísicos , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/agonistas , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cobre/farmacologia , Ácido Glutâmico/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Ratos Transgênicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oligoelementos/farmacologiaRESUMO
The ion-channel TRPV1 is believed to be a major sensor of noxious heat, but surprisingly animals lacking TRPV1 still display marked responses to elevated temperature. In this study, we explored the role of TRPV1-expressing neurons in somatosensation by generating mice wherein this lineage of cells was selectively labelled or ablated. Our data show that TRPV1 is an embryonic marker of many nociceptors including all TRPV1- and TRPM8-neurons as well as many Mrg-expressing neurons. Mutant mice lacking these cells are completely insensitive to hot or cold but in marked contrast retain normal touch and mechanical pain sensation. These animals also exhibit defective body temperature control and lose both itch and pain reactions to potent chemical mediators. Together with previous cell ablation studies, our results define and delimit the roles of TRPV1- and TRPM8-neurons in thermosensation, thermoregulation and nociception, thus significantly extending the concept of labelled lines in somatosensory coding.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Nociceptores/metabolismo , Canais de Cátion TRPV/metabolismo , Termorreceptores/metabolismo , Animais , Temperatura Corporal , Regulação da Temperatura Corporal/genética , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Camundongos , Camundongos Mutantes , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Medição da Dor , Receptores Acoplados a Proteínas G/metabolismo , Teste de Desempenho do Rota-Rod , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Nitrous oxide (N2O, laughing gas) has been used as an anaesthetic and analgesic for almost two centuries, but its cellular targets remain unclear. Here, we present a molecular mechanism of nitrous oxide's selective inhibition of CaV3.2 low-voltage-activated (T-type) calcium channels in pain pathways. Using site-directed mutagenesis and metal chelators such as diethylenetriamine pentaacetic acid and deferoxamine, we reveal that a unique histidine at position 191 of CaV3.2 participates in a critical metal binding site, which may in turn interact with N2O to produce reactive oxygen species (ROS). These free radicals are then likely to oxidize H191 of CaV3.2 in a localized metal-catalysed oxidation reaction. Evidence of hydrogen peroxide and free radical intermediates is given in that N2O inhibition of CaV3.2 channels is attenuated when H2O2 is neutralized by catalase. We also use the adrenochrome test as an indicator of ROS in vitro in the presence of N2O and iron. Ensuing in vivo studies indicate that mice lacking CaV3.2 channels display decreased analgesia to N2O in response to formalin-induced inflammatory pain. Furthermore, a superoxide dismutase and catalase mimetic, EUK-134, diminished pain responses to formalin in wild-type mice, but EUK-134 and N2O analgesia were not additive. This suggests that reduced ROS levels led to decreased inflammation, but without the presence of ROS, N2O was not able to provide additional analgesia. These findings reveal a novel mechanism of interaction between N2O and ion channels, furthering our understanding of this widely used analgesic in pain processing.
Assuntos
Analgésicos não Narcóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Óxido Nitroso/farmacologia , Dor/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adrenocromo/metabolismo , Animais , Canais de Cálcio Tipo T/metabolismo , Catalase/metabolismo , Quelantes/farmacologia , Desferroxamina/farmacologia , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Células HEK293 , Histidina , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Compostos Organometálicos/farmacologia , Oxirredução , Dor/metabolismo , Ácido Pentético/farmacologia , Ratos , Ratos Sprague-Dawley , Salicilatos/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
The effects of anesthetics and analgesics on ion channels have been the subject of intense research since recent reports of direct actions of anesthetic molecules on ion channel proteins. It is now known that ligand-gated channels, particularly γ-amino-butyric acid (GABAA) and N-methyl-D-aspartate (NMDA) receptors, play a key role in mediating anesthetic actions, but these channels are unable to account for all aspects of clinical anesthesia such as loss of consciousness, immobility, analgesia, amnesia, and muscle relaxation. Furthermore, an assortment of voltage-gated and background channels also display anesthetic sensitivity and a key question arises: What role do these other channels play in clinical anesthesia? These channels have overlapping physiological roles and pharmacological profiles, making it difficult to assign aspects of the anesthetic state to individual channel types. Here, we will focus on the function of neuronal voltage-gated calcium channels in mediating the effects of general anesthetics.
Assuntos
Anestésicos Gerais/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Humanos , Potenciais da Membrana , Neurônios/metabolismoRESUMO
Recent studies indicate that T-type calcium channels (T-channels) in the thalamus are cellular targets for general anesthetics. Here, we recorded T-currents and underlying low-threshold calcium spikes from neurons of nucleus reticularis thalami (nRT) in brain slices from young rats and investigated the mechanisms of their modulation by an anesthetic alcohol, 1-octanol. We found that 1-octanol inhibited native T-currents at subanesthetic concentrations with an IC(50) of approximately 4 muM. In contrast, 1-octanol was up to 30-fold less potent in inhibiting recombinant Ca(V)3.3 T-channels heterologously expressed in human embryonic kidney cells. Inhibition of both native and recombinant T-currents was accompanied by a hyperpolarizing shift in steady-state inactivation, indicating that 1-octanol stabilized inactive states of the channel. To explore the mechanisms underlying higher 1-octanol potency in inhibiting native nRT T-currents, we tested the effect of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and PKC inhibitors. We found that PMA caused a modest increase of T-current, whereas the inactive PMA analog 4alpha-PMA failed to affect T-current in nRT neurons. In contrast, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go 6976), an inhibitor of calcium-dependent PKC, decreased baseline T-current amplitude in nRT cells and abolished the effects of subsequently applied 1-octanol. The effects of 1-octanol were also abolished by chelation of intracellular calcium ions with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Taken together, these results suggest that inhibition of calcium-dependent PKC signaling is a possible molecular substrate for modulation of T-channels in nRT neurons by 1-octanol.
Assuntos
1-Octanol/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Cálcio/metabolismo , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Tálamo/citologia , Anestésicos , Animais , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular , Humanos , Concentração Inibidora 50 , RatosRESUMO
Earlier, we showed that streptozocin (STZ)-induced type 1 diabetes in rats leads to the development of painful peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia and mechanical allodynia accompanied by significant enhancement of T-type calcium currents (T-currents) and cellular excitability in medium-sized dorsal root ganglion (DRG) neurons. Here, we studied the in vivo and in vitro effects of gene-silencing therapy specific for the Ca(V)3.2 isoform of T-channels, on thermal and mechanical hypersensitivities, and T-current expression in small- and medium-sized DRG neurons of STZ-treated rats. We found that silencing of the T-channel Ca(V)3.2 isoform using antisense oligonucleotides, had a profound and selective anti-hyperalgesic effect in diabetic rats and is accompanied by significant down-regulation of T-currents in DRG neurons. Anti-hyperalgesic effects of Ca(V)3.2 antisense oligonucleotides in diabetic rats were similar in models of rapid and slow onset of hyperglycemia following intravenous and intraperitoneal injections of STZ, respectively. Furthermore, treatments of diabetic rats with daily insulin injections reversed T-current alterations in DRG neurons in parallel with reversal of thermal and mechanical hypersensitivities in vivo. This confirms that Ca(V)3.2 T-channels, important signal amplifiers in peripheral sensory neurons, may contribute to the cellular hyperexcitability that ultimately leads to the development of painful PDN.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Neuropatias Diabéticas/complicações , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Limiar da Dor/fisiologia , Animais , Canais de Cálcio Tipo T/genética , Neuropatias Diabéticas/induzido quimicamente , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Hiperalgesia/classificação , Hiperalgesia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Insulina/uso terapêutico , Potenciais da Membrana/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Limiar da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Estreptozocina , Fatores de TempoRESUMO
Alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid; lipoic acid) is an endogenous compound used to treat pain disorders in humans, but its mechanisms of analgesic action are not well understood. Here, we show that lipoic acid selectively inhibited native Ca(V)3.2 T-type calcium currents (T-currents) and diminished T-channel-dependent cellular excitability in acutely isolated rat sensory neurons. Lipoic acid locally injected into peripheral receptive fields of pain-sensing sensory neurons (nociceptors) in vivo decreased sensitivity to noxious thermal and mechanical stimuli in wild-type but not Ca(V)3.2 knock-out mice. Ensuing molecular studies demonstrated that lipoic acid inhibited recombinant Ca(V)3.2 channels heterologously expressed in human embryonic kidney 293 cells by oxidating specific thiol residues on the cytoplasmic face of the channel. This study provides the first mechanistic demonstration of a nociceptive ion channel modulation that may contribute to the documented analgesic properties of lipoic acid in vivo.
Assuntos
Analgésicos/farmacologia , Canais de Cálcio Tipo T/metabolismo , Dor/tratamento farmacológico , Ácido Tióctico/farmacologia , Ácido Tióctico/fisiologia , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo T/genética , Linhagem Celular , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Oxirredução , Dor/metabolismo , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismoRESUMO
Previous data have indicated that T-type calcium channels (low-voltage activated T-channels) are potently inhibited by volatile anesthetics. Although the interactions of T-channels with a number of anesthetics have been described, the mechanisms by which these agents modulate channel activity, and the functional consequences of such interactions, are not well studied. Here, we used patch-clamp recordings to explore the actions of a prototypical volatile anesthetic, isoflurane (Iso), on recombinant human Ca(V)3.1 and Ca(V)3.2 isoforms of T-channels. We also performed behavioral testing of anesthetic endpoints in mice lacking Ca(V)3.2. Iso applied at resting channel states blocked current through both isoforms in a similar manner at clinically relevant concentrations (1 minimum alveolar concentration, MAC). Inhibition was more prominent at depolarized membrane potentials (-65 versus -100 mV) as evidenced by hyperpolarizing shifts in channel availability curves and a 2.5-fold decrease in IC(50) values. Iso slowed recovery from inactivation and enhanced deactivation in both Ca(V)3.1 and Ca(V)3.2 in a comparable manner but caused a depolarizing shift in activation curves and greater use-dependent block of Ca(V)3.2 channels. In behavioral tests, Ca(V)3.2 knockout (KO) mice showed significantly decreased MAC in comparison with wild-type (WT) litter mates. KO and WT mice did not differ in loss of righting reflex, but mutant mice displayed a delayed onset of anesthetic induction. We conclude that state-dependent inhibition of T-channel isoforms in the central and peripheral nervous systems may contribute to isoflurane's important clinical effects.