RESUMO
The vanilloid receptor 1 (TRPV1) is activated by capsaicin, several endogenous lipids, acidic pH and elevated temperatures. Inflammatory mediators (BK, substance P) also modulate TRPV1 activity. In this study we investigated the effect of TRPV1 agonists and elevated temperatures on neuronal membrane excitability by electrophysiological techniques using freshly isolated rat dorsal root ganglion neurons (DRGs). Focal application of heated solutions demonstrated that the normal threshold (approximately 42 degrees C) of TRPV1 activation was reduced in the presence of capsaicin (1 microM) to approximately 30 degrees C. In current-clamp recordings, increasing the temperature of the solution resulted in larger membrane depolarizations and significantly altered the pattern and onset of the action potential train evoked by 1 microM capsaicin. These effects were blocked by the TRPV1 antagonist capsazepine (10 microM). In contrast to capsaicin, anandamide (10 microM) alone did not evoke action potentials, but it did alter the excitability of neurons to subsequent applications of heat (50 degrees C). Together these results provide evidence that a synergistic interaction of TRPV1 ligands and elevated temperature activates TRPV1 receptors and results in profound effects on membrane excitability.
Assuntos
Gânglios Espinais/fisiologia , Canais de Cátion TRPV/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Ácidos Araquidônicos/farmacologia , Capsaicina/farmacologia , Endocanabinoides , Gânglios Espinais/efeitos dos fármacos , Masculino , Fosforilação , Alcamidas Poli-Insaturadas/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/agonistas , TemperaturaRESUMO
Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N'-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.
Assuntos
Analgésicos/farmacocinética , Sistema Nervoso Central/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Dor/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Administração Oral , Analgésicos/metabolismo , Animais , Artralgia/tratamento farmacológico , Artralgia/metabolismo , Artralgia/fisiopatologia , Cálcio/metabolismo , Capsaicina/antagonistas & inibidores , Linhagem Celular , Células Cultivadas , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Indazóis/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Injeções Espinhais , Masculino , Nociceptores/metabolismo , Dor/metabolismo , Dor/fisiopatologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonas/farmacologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Resultado do Tratamento , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
1. Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small-conductance Ca2+-activated K+ channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole-cell patch clamp configuration. 2. Depolarization activated a K+-selective, apamin-sensitive and iberiotoxin-insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mM EGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+-activated SK channels expressed only in denervated muscle. 3. Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+ due to release from the sarcoplasmic reticulum (SR) and influx through voltage-gated Ca2+ channels. 4. The envelope of SK tail currents was diminished by 10 microM ryanodine for all pulse durations, whereas 2 mM cobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+ release from the SR during short pulses primarily activated SK channels. 5. In current clamp mode with the resting membrane potential set at -70 mV, denervation decreased the action potential threshold by approximately 8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold. 6. These results are consistent with a model in which SK channel activity in the T-tubules of denervated skeletal muscle causes a local increase in K+ concentration that results in hyperexcitability.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Apamina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Quelantes/farmacologia , Cobalto/farmacologia , Ácido Egtázico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Denervação Muscular , Músculo Esquelético/citologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Potássio/metabolismo , Retículo Sarcoplasmático/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância BaixaRESUMO
The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at -80 mV, barium currents (I(Ba)s) were evoked by voltage commands to above -35 mV that peaked at +5 mV. When holding potentials were reduced to -20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I(Ba) was fit with a Boltzmann curve (V(1/2) = -43.3 mV; slope = -17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I(Ba)s were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 microM nifedipine (19 vs. 25%), 10 microM conotoxin GVIA (27 vs. 25%), 10 microM conotoxin MVIIC (15 vs. 16%), and 1.75 microM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 microM cadmium. In the presence of nifedipine, GVIA, and MVIIC, approximately 35% of current remained, which was reduced further by SNX-482 (7-14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).
Assuntos
Canais de Cálcio/biossíntese , Neurônios/metabolismo , Teratocarcinoma/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/biossíntese , Canais de Cálcio Tipo N/biossíntese , Canais de Cálcio Tipo P/biossíntese , Canais de Cálcio Tipo Q/biossíntese , Canais de Cálcio Tipo R/biossíntese , Diferenciação Celular , Relação Dose-Resposta a Droga , Humanos , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Venenos de Aranha/farmacologia , Teratocarcinoma/patologia , Células Tumorais Cultivadas , ômega-Conotoxina GVIA/farmacologia , ômega-Conotoxinas/farmacologiaRESUMO
During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.
Assuntos
Neurônios/química , Receptores de GABA-A/análise , Teratocarcinoma/química , Ansiolíticos/uso terapêutico , Anticonvulsivantes/uso terapêutico , Diferenciação Celular/fisiologia , Diazepam/uso terapêutico , Furosemida/uso terapêutico , Humanos , Lantânio/uso terapêutico , Neurônios/patologia , Técnicas de Patch-Clamp , Piridinas/uso terapêutico , Teratocarcinoma/patologia , Triazóis/uso terapêutico , Células Tumorais Cultivadas , Zinco/uso terapêutico , ZolpidemRESUMO
mRNA encoding the recently cloned gamma-aminobuytyric acid(A) receptor (GABAR) pi subunit is expressed in the hippocampus and in several non-neuronal tissues including the uterus and ovaries. Whereas native GABARs are pentamers composed primarily of alphabetagamma, alphabetadelta, or alphabetaepsilon subunits, it has not been demonstrated clearly that the pi subunit incorporates into functional GABARs to form alphabetapi receptors and, if so, with what properties. We provide electrophysiological evidence that the pi subunit can coassemble with either alpha5beta3 or alpha5beta3gamma3 subunits to produce recombinant GABARs with distinct pharmacological and biophysical properties. Compared with alpha5beta3 receptors, GABARs produced by coexpression of alpha5beta3pi subunits had a lower GABA EC(50) value, were enhanced to a lesser extent by loreclezole, had different IC(50) values for pregnenolone sulfate and lanthanum, and were insensitive to benzodiazepines. Incorporation of both pi and gamma3 subunits into an alpha5beta3gamma3pi isoform was suggested by reduced enhancement by diazepam and a high zinc IC(50) value. Current-voltage relations for the alpha5beta3pi subunit combination outwardly rectified more than currents from alpha5beta3gamma3 but less than alpha5beta3 combination GABARs. Single-channel alpha5beta3 GABAR currents had a main conductance state of 15.2 picoSeimens (pS). Coexpression of the pi subunit with alpha5beta3 subtypes increased the conductance level to 23.8 pS, similar to the conductance level of alpha5beta3gamma3 GABARs (26.9 pS). We conclude that the pi subunit coassembles with alpha, beta, and gamma subunits to form functional alphabetapi or alphabetagammapi GABARs and, thus, could have a significant impact on the function of native GABARs expressed in the brain or non-neuronal tissue.
Assuntos
Receptores de GABA-A/metabolismo , Animais , Barbitúricos/farmacologia , Benzodiazepinas/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Eletrofisiologia , Lantânio/farmacologia , Camundongos , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esteroides/farmacologia , Triazóis/farmacologia , Zinco/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
A new gamma-aminobutyric acid (GABA)A receptor (GABAR) subunit class, epsilon, has recently been cloned and shown to form functional channels when coexpressed with both alpha and beta subunits. We report that the combination of alpha1beta3epsilon subunit subtypes expressed in L929 cells produced functional chloride ion channels that were both spontaneously active and gated by the application of extracellular GABA. When cells were voltage-clamped at -75 mV in the whole-cell configuration, holding currents of 50 to 300 pA associated with increased noise were consistently recorded. The application of pentobarbital and loreclezole, which increase GABAR currents, increased the holding current, whereas the application of zinc and picrotoxin, which reduce GABAR currents, reduced the holding current in a concentration-dependent manner. Coexpression of alpha1beta3gamma2L, alpha1beta3delta, alpha1epsilon, beta3epsilon, alpha1beta3, or epsilon subtypes did not produce holding currents that were sensitive to picrotoxin (30 microM). Cells expressing alpha1 beta3epsilon subtypes had concentration-dependent GABAR currents that were potentiated by pentobarbital, loreclezole, and lanthanum and inhibited by zinc and furosemide. Spontaneous and GABAR single-channel currents from alpha1beta3epsilon receptors had single-channel conductances of approximately 24 pS. The biophysical properties and the effects of allosteric modulators were similar for spontaneous and evoked GABAR currents, suggesting that a single GABAR isoform was responsible for both currents. These data extend the pharmacological characterization of epsilon-containing GABARs and demonstrate that incorporation of the epsilon subunit permits spontaneous channel gating while preserving the structural information necessary for GABA sensitivity.
Assuntos
Receptores de GABA-A/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Furosemida/farmacologia , Lantânio/farmacologia , Camundongos , Pentobarbital/farmacologia , Isoformas de Proteínas/efeitos dos fármacos , Ratos , Receptores de GABA-A/fisiologia , Triazóis/farmacologia , Zinco/farmacologiaRESUMO
Human NT2 teratocarcinoma cells differentiate into neuron-like NT2-N cells when treated with retinoic acid. GABA evoked concentration-dependent whole-cell currents in NT2-N cells with an EC50 of 21.8 microM and a Hill slope of 1.2. GABAA receptor (GABAR) currents reversed at ECl- and did not display voltage-dependent rectification. GABAR single channels opened in bursts to a 23 pS main conductance level and a 19 pS subconductance level, with infrequent openings to a 27 pS conductance level. Kinetic properties of the main conductance level were similar to other native and recombinant GABAR channels. Diazepam and zolpidem enhanced GABAR currents with moderate affinity, whereas methyl-6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate inhibited GABAR currents. Loreclezole enhanced GABAR currents with high affinity, but furosemide antagonized GABAR currents with low affinity. The neurosteroids alphaxalone and pregnenolone sulfate appropriately modulated GABAR currents. Zinc blocked GABAR currents with low affinity, but lanthanum did not significantly alter NT2-N GABAR currents. Reverse transcription PCR (RT-PCR) performed on RNA from NT2-N cells clearly detected transcripts encoding human alpha2, alpha3, alpha5, beta3, gamma3, and pi subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an alpha2 and/or alpha3 subtype combined with the beta3 and gamma3 subtypes. The data do not rule out receptors containing combinations of alpha2 and/or alpha3 subtypes with the alpha5 subtype or receptors with both beta1 and beta3 subtypes. The presence or absence or the pi subunit in functionally expressed receptors could not be determined.
Assuntos
Neurônios/química , Receptores de GABA-A , Anticonvulsivantes/farmacologia , Bicuculina/farmacologia , Carbolinas/farmacologia , Convulsivantes/farmacologia , Primers do DNA , Diazepam/farmacologia , Diuréticos/farmacologia , Furosemida/farmacologia , Moduladores GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Humanos , Hipnóticos e Sedativos/farmacologia , Lantânio/farmacologia , Neurônios/citologia , Técnicas de Patch-Clamp , Pentobarbital/farmacologia , Fenobarbital/farmacologia , Picrotoxina/farmacologia , Reação em Cadeia da Polimerase , Pregnanodionas/farmacologia , Pregnenolona/farmacologia , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Teratocarcinoma , Triazóis/farmacologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologia , Zinco/farmacologia , Zolpidem , Ácido gama-Aminobutírico/farmacologiaRESUMO
Functional studies have indicated that, unlike most divalent cations, lanthanum increases both native and recombinant gamma-aminobutyric acid (GABA) receptor (GABAR) currents. In the present study, we have examined whether lanthanum shows subunit-dependent selectivity for modification of currents from different GABAR isoforms. The effects of lanthanum on three different GABAR isoforms, alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta, were determined by transient expression of combinations of alpha1, alpha6, beta3, gamma2L, and delta subunit cDNAs in L929 fibroblasts. Whole-cell recording was used to determine the concentration-response curves for lanthanum for the three different isoforms at submaximal concentrations of GABA. Lanthanum displayed strong potentiation of alpha1beta3gamma2L GABAR currents consistent with earlier reports of potentiation of GABAR currents by lanthanum in neurons and recombinant GABAR isoforms. However, in contrast to the potentiation of alpha1beta3gamma2L GABAR currents by lanthanum, alpha6beta3delta GABAR currents were strongly inhibited and alpha6beta3gamma2L GABAR currents were weakly inhibited by lanthanum. Interaction of lanthanum with GABAR isoforms was competitive, with lanthanum decreasing the EC50 value for GABA of alpha1beta3gamma2L GABARs without changing the maximum current and increasing the EC50 value for GABA of alpha6beta3delta and alpha6beta3gamma2L GABAR currents (greater shift in EC50 value in the alpha6beta3delta compared with the alpha6beta3gamma2L GABARs) without changing the maximum GABAR current. Neither potentiation nor inhibition of GABAR currents by lanthanum showed any voltage dependence. These results suggest that 1) changing the alpha-subunit subtype from alpha1 to alpha6 altered the effect of lanthanum from potentiation to inhibition, 2) changing the gamma2L subunit to the delta-subunit changed the level of maximal inhibition of alpha6 subtype-containing GABAR currents by lanthanum, and 3) the site for interaction with lanthanum probably was on the extracellular surface of GABARs.
Assuntos
Fibroblastos/metabolismo , Lantânio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Receptores de GABA/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Técnicas de Patch-ClampRESUMO
Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.
Assuntos
Separação Imunomagnética/métodos , Receptores de GABA-A/imunologia , Animais , Linhagem Celular , Camundongos , Técnicas de Patch-Clamp , Receptores de GABA-A/efeitos dos fármacos , Transfecção/métodos , Ácido gama-Aminobutírico/farmacologiaRESUMO
The cDNAs encoding alpha 5 and gamma 2L subunit subtypes of the gamma-aminobutyric acid (GABA) type A receptor (GABAR) were transfected into L929 cells together with cDNAs encoding either the beta 1, beta 2, or beta 3 subunit subtype. Properties of expressed recombinant alpha 5 beta X gamma 2L (where X = 1,2, or 3) GABARs were studied with the use of whole-cell, patch-clamp techniques. In cells voltage-clamped at -70 mV with equlvalent bath and pipette chloride concentrations, the application of GABA produced a concentration-dependent inward chloride current with all three alpha 5 beta X gamma 2L isoforms. Minimal or no responses were recorded from cells transfected with only two subunit cDNAs, demonstrating that all three subunits were required for functional receptor assembly in these cells. The GABA concentration producing a half-maximal current was similar for beta 2 and beta 3 subtype-containing receptors (6 microM) but higher for beta 1 subtype-containing receptors (26 microM). alpha 5 beta 3 gamma 2L receptors were zinc and diazepam sensitive but zolpidem insensitive. In response to low GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed outward rectification of the current-voltage relationship, whereas current-voltage responses of beta 2 subtype-containing receptors were relatively linear. Likewise, at high GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed less desensitization at positive than at negative membrane potentials. Beta 2 subtype-containing receptors displayed faster desensitization at depolarized potentials. These voltage-dependent properties were characteristic of alpha 5 but not alpha 1 or alpha 6 subtype-containing receptors and were similar to responses recorded from hippocampal CA1 pyramidal neurons. Based on the pharmacological and biophysical similarities to hippocampal GABAR responses, the alpha 5 beta 3 gamma 2L isoform could represent a native GABAR subtype.