RESUMO
Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five- to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.
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Volatile anesthetics depress neurotransmitter release in a brain region- and neurotransmitter-selective manner by unclear mechanisms. Voltage-gated sodium channels (Navs), which are coupled to synaptic vesicle exocytosis, are inhibited by volatile anesthetics through reduction of peak current and modulation of gating. Subtype-selective effects of anesthetics on Nav might contribute to observed neurotransmitter-selective anesthetic effects on release. We analyzed anesthetic effects on Na+ currents mediated by the principal neuronal Nav subtypes Nav1.1, Nav1.2, and Nav1.6 heterologously expressed in ND7/23 neuroblastoma cells using whole-cell patch-clamp electrophysiology. Isoflurane at clinically relevant concentrations induced a hyperpolarizing shift in the voltage dependence of steady-state inactivation and slowed recovery from fast inactivation in all three Nav subtypes, with the voltage of half-maximal steady-state inactivation significantly more positive for Nav1.1 (-49.7 ± 3.9 mV) than for Nav1.2 (-57.5 ± 1.2 mV) or Nav1.6 (-58.0 ± 3.8 mV). Isoflurane significantly inhibited peak Na+ current (I Na) in a voltage-dependent manner: at a physiologically relevant holding potential of -70 mV, isoflurane inhibited peak I Na of Nav1.2 (16.5% ± 5.5%) and Nav1.6 (18.0% ± 7.8%), but not of Nav1.1 (1.2% ± 0.8%). Since Nav subtypes are differentially expressed both between neuronal types and within neurons, greater inhibition of Nav1.2 and Nav1.6 compared with Nav1.1 could contribute to neurotransmitter-selective effects of isoflurane on synaptic transmission.
Assuntos
Anestésicos Gerais/farmacologia , Isoflurano/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Cinética , Neurônios/metabolismo , Neurotransmissores/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Voltage-gated Na+ channels (Nav ) modulate neuronal excitability, but the roles of the various Nav subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Nav subtypes (Nav 1.1, Nav 1.2, Nav 1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Nav subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre- and post-synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Nav 1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Nav 1.2 and Nav 1.6 were expressed both pre- and post-synaptically. In contrast, in the stratum radiatum (SR) of CA1, Nav 1.1, Nav 1.2, and Nav 1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Nav subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Nav 1.1 and Nav 1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Nav 1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Nav subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Nav expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.
Assuntos
Hipocampo/citologia , Neurônios/fisiologia , Densidade Pós-Sináptica/metabolismo , Terminações Pré-Sinápticas/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Células Cultivadas , Embrião de Mamíferos , Células HEK293 , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/ultraestrutura , Plasticidade Neuronal/genética , Neurônios/ultraestrutura , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/ultraestrutura , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/ultraestruturaRESUMO
General anesthetics revolutionized medicine by allowing surgeons to perform more complex and much longer procedures. This widely used class of drugs is essential to patient care, yet their exact molecular mechanism(s) are incompletely understood. One early hypothesis over a century ago proposed that nonspecific interactions of anesthetics with the lipid bilayer lead to changes in neuronal function via effects on membrane properties. This model was supported by the Meyer-Overton correlation between anesthetic potency and lipid solubility and despite more recent evidence for specific protein targets, in particular ion-channels, lipid bilayer-mediated effects of anesthetics is still under debate. We therefore tested a wide range of chemically diverse general anesthetics on lipid bilayer properties using a sensitive and functional gramicidin-based assay. None of the tested anesthetics altered lipid bilayer properties at clinically relevant concentrations. Some anesthetics did affect the bilayer, though only at high supratherapeutic concentrations, which are unlikely relevant for clinical anesthesia. These results suggest that anesthetics directly interact with membrane proteins without altering lipid bilayer properties at clinically relevant concentrations. Voltage-gated Na+ channels are potential anesthetic targets and various isoforms are inhibited by a wide range of volatile anesthetics. They inhibit channel function by reducing peak Na+ current and shifting steady-state inactivation toward more hyperpolarized potentials. Recent advances in crystallography of prokaryotic Na+ channels, which are sensitive to volatile anesthetics, together with molecular dynamics simulations and electrophysiological studies will help identify potential anesthetic interaction sites within the channel protein itself.
Assuntos
Anestésicos Gerais/farmacologia , Bicamadas Lipídicas/metabolismo , Canais de Sódio/metabolismo , Animais , Gramicidina/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Voltage-gated Na+ channels (Nav) have emerged as important presynaptic targets for volatile anesthetic (VA) effects on synaptic transmission. However, the detailed biophysical mechanisms by which VAs modulate Nav function remain unclear. VAs alter macroscopic activation and inactivation of the prokaryotic Na+ channel, NaChBac, which provides a useful structural and functional model of mammalian Nav Here, we study the effects of the common general anesthetic isoflurane on NaChBac function by analyzing macroscopic Na+ currents (INa) in wild-type (WT) channels and mutants with impaired (G229A) or enhanced (G219A) inactivation. We use a previously described six-state Markov model to analyze empirical WT and mutant NaChBac channel gating data. The model reproduces the mean empirical gating manifest in INa time courses and optimally estimates microscopic rate constants, valences (z), and fractional electrical distances (x) of forward and backward transitions. The model also reproduces gating observed for all three channels in the absence or presence of isoflurane, providing further validation. We show using this model that isoflurane increases forward activation and inactivation rate constants at 0 mV, which are associated with estimated chemical free energy changes of approximately -0.2 and -0.7 kcal/mol, respectively. Activation is voltage dependent (z ≈ 2e0, x ≈ 0.3), inactivation shows little voltage dependence, and isoflurane has no significant effect on either. Forward inactivation rate constants are more than 20-fold greater than backward rate constants in the absence or presence of isoflurane. These results indicate that isoflurane modulates NaChBac gating primarily by increasing forward activation and inactivation rate constants. These findings support accumulating evidence for multiple sites of anesthetic interaction with the channel.
Assuntos
Anestésicos Inalatórios/farmacologia , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Isoflurano/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células HEK293 , Humanos , Domínios Proteicos , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
General anesthetics have revolutionized medicine by facilitating invasive procedures, and have thus become essential drugs. However, detailed understanding of their molecular mechanisms remains elusive. A mechanism proposed over a century ago involving unspecified interactions with the lipid bilayer known as the unitary lipid-based hypothesis of anesthetic action, has been challenged by evidence for direct anesthetic interactions with a range of proteins, including transmembrane ion channels. Anesthetic concentrations in the membrane are high (10-100 mM), however, and there is no experimental evidence ruling out a role for the lipid bilayer in their ion channel effects. A recent hypothesis proposes that anesthetic-induced changes in ion channel function result from changes in bilayer lateral pressure that arise from partitioning of anesthetics into the bilayer. We examined the effects of a broad range of chemically diverse general anesthetics and related nonanesthetics on lipid bilayer properties using an established fluorescence assay that senses drug-induced changes in lipid bilayer properties. None of the compounds tested altered bilayer properties sufficiently to produce meaningful changes in ion channel function at clinically relevant concentrations. Even supra-anesthetic concentrations caused minimal bilayer effects, although much higher (toxic) concentrations of certain anesthetic agents did alter lipid bilayer properties. We conclude that general anesthetics have minimal effects on bilayer properties at clinically relevant concentrations, indicating that anesthetic effects on ion channel function are not bilayer-mediated but rather involve direct protein interactions.
Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Bicamadas Lipídicas/química , Gramicidina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Estrutura MolecularRESUMO
Although general anesthetics are clinically important and widely used, their molecular mechanisms of action remain poorly understood. Volatile anesthetics such as isoflurane (ISO) are thought to alter neuronal function by depressing excitatory and facilitating inhibitory neurotransmission through direct interactions with specific protein targets, including voltage-gated sodium channels (Na(v)). Many anesthetics alter lipid bilayer properties, suggesting that ion channel function might also be altered indirectly through effects on the lipid bilayer. We compared the effects of ISO and of a series of fluorobenzene (FB) model volatile anesthetics on Na(v) function and lipid bilayer properties. We examined the effects of these agents on Na(v) in neuronal cells using whole-cell electrophysiology, and on lipid bilayer properties using a gramicidin-based fluorescence assay, which is a functional assay for detecting changes in lipid bilayer properties sensed by a bilayer-spanning ion channel. At clinically relevant concentrations (defined by the minimum alveolar concentration), both the FBs and ISO produced prepulse-dependent inhibition of Na(v) and shifted the voltage dependence of inactivation toward more hyperpolarized potentials without affecting lipid bilayer properties, as sensed by gramicidin channels. Only at supra-anesthetic (toxic) concentrations did ISO alter lipid bilayer properties. These results suggest that clinically relevant concentrations of volatile anesthetics alter Na(v) function through direct interactions with the channel protein with little, if any, contribution from changes in bulk lipid bilayer properties. Our findings further suggest that changes in lipid bilayer properties are not involved in clinical anesthesia.
Assuntos
Isoflurano/administração & dosagem , Isoflurano/química , Bicamadas Lipídicas/química , Sódio/química , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/fisiologia , Anestésicos Inalatórios/administração & dosagem , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Fluorbenzenos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Camundongos , Neurônios , Bloqueadores dos Canais de Sódio/administração & dosagem , Compostos Orgânicos Voláteis/administração & dosagemRESUMO
General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However, the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines, a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D, drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus, isoflurane destabilizes spine F-actin, resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus, a locus for anesthetic-induced amnesia, and have important implications for anesthetic effects on synaptic plasticity.
Assuntos
Actinas/metabolismo , Anestésicos Inalatórios/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Isoflurano/farmacologia , Animais , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , RatosRESUMO
A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.
Assuntos
Membrana Celular/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Proteínas de Membrana/fisiologia , Simulação de Dinâmica MolecularRESUMO
Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.
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Anestésicos Intravenosos/farmacologia , Anestésicos/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Canais de Potássio/efeitos dos fármacos , Propofol/análogos & derivados , Propofol/farmacologia , Algoritmos , Anestésicos/uso terapêutico , Anestésicos Intravenosos/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Disponibilidade Biológica , DNA Complementar/biossíntese , DNA Complementar/genética , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Temperatura Alta , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Indicadores e Reagentes , Bicamadas Lipídicas , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Propofol/uso terapêutico , XenopusRESUMO
The molecular mechanisms of modern inhaled anesthetics are still poorly understood although they are widely used in clinical settings. Considerable evidence supports effects on membrane proteins including ligand- and voltage-gated ion channels of excitable cells. Na(+) channels are crucial to action potential initiation and propagation, and represent potential targets for volatile anesthetic effects on central nervous system depression. Inhibition of presynaptic Na(+) channels leads to reduced neurotransmitter release at the synapse and could therefore contribute to the mechanisms by which volatile anesthetics produce their characteristic end points: amnesia, unconsciousness, and immobility. Early studies on crayfish and squid giant axon showed inhibition of Na(+) currents by volatile anesthetics at high concentrations. Subsequent studies using native neuronal preparations and heterologous expression systems with various mammalian Na(+) channel isoforms implicated inhibition of presynaptic Na(+) channels in anesthetic actions at clinical concentrations. Volatile anesthetics reduce peak Na(+) current (I(Na)) and shift the voltage of half-maximal steady-state inactivation (h(∞)) toward more negative potentials, thus stabilizing the fast-inactivated state. Furthermore recovery from fast-inactivation is slowed, together with enhanced use-dependent block during pulse train protocols. These effects can depress presynaptic excitability, depolarization and Ca(2+) entry, and ultimately reduce transmitter release. This reduction in transmitter release is more potent for glutamatergic compared to GABAergic terminals. Involvement of Na(+) channel inhibition in mediating the immobility caused by volatile anesthetics has been demonstrated in animal studies, in which intrathecal infusion of the Na(+) channel blocker tetrodotoxin increases volatile anesthetic potency, whereas infusion of the Na(+) channels agonist veratridine reduces anesthetic potency. These studies indicate that inhibition of presynaptic Na(+) channels by volatile anesthetics is involved in mediating some of their effects.
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The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator-activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer-water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs' partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer-dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.
Assuntos
Insulina/metabolismo , Bicamadas Lipídicas/metabolismo , Canais de Sódio/metabolismo , Tiazolidinedionas/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromanos/farmacologia , Descoberta de Drogas/métodos , Gramicidina/metabolismo , Canais Iônicos/fisiologia , Bicamadas Lipídicas/química , Potenciais da Membrana/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , PPAR gama/metabolismo , Pioglitazona , Ratos , Rosiglitazona , Tiazolidinedionas/química , Tiazolidinedionas/metabolismo , TroglitazonaRESUMO
BACKGROUND: Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear. METHODS: The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology. RESULTS: From a holding potential of -70 mV, isoflurane (0.53 +/- 0.06 mM, 1.8 minimum alveolar concentration at 24 degrees C) reduced normalized peak Na current (INa) of Nav1.8 to 0.55 +/- 0.03 and of endogenous TTX-s Nav to 0.56 +/- 0.06. Isoflurane minimally inhibited INa from a holding potential of -140 mV. Isoflurane did not affect voltage-dependence of activation, but it significantly shifted voltage-dependence of steady-state inactivation by -6 mV for Nav1.8 and by -7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67 +/- 0.06 mM for Nav1.8 and 0.66 +/- 0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 minimum alveolar concentration. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56 +/- 0.08 mM isoflurane reduced INa to 0.64 +/- 0.01 versus 0.78 +/- 0.01 for control. CONCLUSION: Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
Assuntos
Anestésicos Inalatórios/farmacologia , Isoflurano/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Resistência a Medicamentos , Estimulação Elétrica , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.8 , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Ratos , Canais de Sódio/genética , TransfecçãoRESUMO
BACKGROUND: Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. METHODS: Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. RESULTS: Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. CONCLUSIONS: Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.