Logicians have worked with so many different logical systems that it is not possible even to esti-mate the number. Of these, many are best seen as extensions of classical logic, including both thoseof interest to math...Logicians have worked with so many different logical systems that it is not possible even to esti-mate the number. Of these, many are best seen as extensions of classical logic, including both thoseof interest to mathematics and those of interest to philosophy and computer science. (Henceforth Iwill use the term "intelligent systems theory" for the common ground of philosophical logic and展开更多
OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues i...OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues in GABAAreceptors(GABAARs) were also investigated.METHODS Using a zebrafish larvae locomotion model,we investigated the detailed dose-dependent effects of diazepam and other BZDs on zebrafish larvae behaviors,with a focus on their high-dose effects.We then evaluated the influence of the classical BZD antagonist flumazenil,GABAARs antagonist bicuculline,and the antagonist of a proposed BZD binding site in α4/6β3δ subtype receptor Ro15-4513 on BZDs induced immobility.Using wholecell patch clamp electrophysiological recordings on recombinant GABAARs,we investigated the modulation of diazepam alone or combined with flumazenil on GABA-elicited current in wildtype and mutated receptors.RESULTS Diazepam dose-dependently decreased the locomotor activities of zebrafish larvae at doses of 0.4,2,10,20,30,50 and 75 mg·L^(-1).The hypolocomotion(sedation-like state) induced by diazepam at10 and 20 mg·L^(-1) were effectively antagonized by flumazenil with EC150 of 0.086 mg·L-and1.295 mg·L^(-1),while the immobility(anesthesialike state) induced by diazepam at 30 mg·L^(-1) was abolished by bicuculline(3 mg·L^(-1)),but not affected by flumazenil(even at concentration up to150 mg·L^(-1)) or Ro15-4513(100 mg·L^(-1)).The immobility induced by clonazepam and lorazepam(100 mg·L^(-1)) was also resistant to flumazenil(100 mg·L^(-1)).In the α1β2γ2 subtype receptor expressed in HEK293 T cells,diazepam dose-dependently potentiated GABA-elicited current,and this potentiation was effectively antagonized by flumazenil(100 μmol·L^(-1)).However,in α1β2 subtype receptor,diazepam(150 μmol·L^(-1)) induced potentiation was insensitive to flumazenil(100 μmol·L^(-1)),but was abolished by the mutation of β2 N265 I.CONCLUSION These results provide direct in vivo evidence for the nonclassical binding sites,which may be located at the second transmembrane domain of GABAAR,mediate BZD-induced anesthesia.展开更多
OBJECTIVE To identify benzodi⁃azepine(BZD)effects that are insensitive to the classical BZD binding site antagonist,flumazenil.Whether the flumazenil-insensitive BZD effects have selectivity on different GABAA recepto...OBJECTIVE To identify benzodi⁃azepine(BZD)effects that are insensitive to the classical BZD binding site antagonist,flumazenil.Whether the flumazenil-insensitive BZD effects have selectivity on different GABAA receptor sub⁃types was also investigated.METHODS The high-concentration effects of BZDs and their sensitivity to flumazenil were determined on recombi⁃nant synaptic(α1β2γ2,α2β2γ2,α5β2γ2)and extra-synaptic(α4β2δ)GABAA receptors using the voltage-clamp electrophysiology technique.The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice loss of right reflex(LORR)test.RESULTS Diazepam induced a biphasic potentiation for theα1β2γ2,α2β2γ2 andα5β2γ2 receptor channels,but did not affect theα4β2δreceptor.In contrast to the nanomolar com⁃ponent of potentiation,the second potentiation elicited by micromolar diazepam was insensitive to flumazenil.Midazolam,clonazepam,and loraz⁃epam at 200μmol·L-1 exhibited similar flumaze⁃nil-insensitive effects onα1β2γ2,α2β2γ2 andα5β2γ2 receptors,whereas the potentiation induced by 200μmol·L-1 zolpidem or triazolam was abol⁃ished by flumazenil.Consistent with the in vitro results,flumazenil antagonized the zolpidem(50 mg·kg-1)-induced LORR,but not those induced by 50 mg·kg-1 diazepam or 100 mg·kg-1 midazolam.CONCLUSION The existence of non-classical BZD binding sites on certain GABAA receptor subtypes and the flumazenil-insensitive effects depend on the chemical structures of the allosteric modulators.展开更多
文摘Logicians have worked with so many different logical systems that it is not possible even to esti-mate the number. Of these, many are best seen as extensions of classical logic, including both thoseof interest to mathematics and those of interest to philosophy and computer science. (Henceforth Iwill use the term "intelligent systems theory" for the common ground of philosophical logic and
基金Foundation for Young Scientists of Beijing Institute of Pharmacology and Toxicology.
文摘OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues in GABAAreceptors(GABAARs) were also investigated.METHODS Using a zebrafish larvae locomotion model,we investigated the detailed dose-dependent effects of diazepam and other BZDs on zebrafish larvae behaviors,with a focus on their high-dose effects.We then evaluated the influence of the classical BZD antagonist flumazenil,GABAARs antagonist bicuculline,and the antagonist of a proposed BZD binding site in α4/6β3δ subtype receptor Ro15-4513 on BZDs induced immobility.Using wholecell patch clamp electrophysiological recordings on recombinant GABAARs,we investigated the modulation of diazepam alone or combined with flumazenil on GABA-elicited current in wildtype and mutated receptors.RESULTS Diazepam dose-dependently decreased the locomotor activities of zebrafish larvae at doses of 0.4,2,10,20,30,50 and 75 mg·L^(-1).The hypolocomotion(sedation-like state) induced by diazepam at10 and 20 mg·L^(-1) were effectively antagonized by flumazenil with EC150 of 0.086 mg·L-and1.295 mg·L^(-1),while the immobility(anesthesialike state) induced by diazepam at 30 mg·L^(-1) was abolished by bicuculline(3 mg·L^(-1)),but not affected by flumazenil(even at concentration up to150 mg·L^(-1)) or Ro15-4513(100 mg·L^(-1)).The immobility induced by clonazepam and lorazepam(100 mg·L^(-1)) was also resistant to flumazenil(100 mg·L^(-1)).In the α1β2γ2 subtype receptor expressed in HEK293 T cells,diazepam dose-dependently potentiated GABA-elicited current,and this potentiation was effectively antagonized by flumazenil(100 μmol·L^(-1)).However,in α1β2 subtype receptor,diazepam(150 μmol·L^(-1)) induced potentiation was insensitive to flumazenil(100 μmol·L^(-1)),but was abolished by the mutation of β2 N265 I.CONCLUSION These results provide direct in vivo evidence for the nonclassical binding sites,which may be located at the second transmembrane domain of GABAAR,mediate BZD-induced anesthesia.
基金Institutional funding from Beijing Institute of Pharmacology and Toxicology。
文摘OBJECTIVE To identify benzodi⁃azepine(BZD)effects that are insensitive to the classical BZD binding site antagonist,flumazenil.Whether the flumazenil-insensitive BZD effects have selectivity on different GABAA receptor sub⁃types was also investigated.METHODS The high-concentration effects of BZDs and their sensitivity to flumazenil were determined on recombi⁃nant synaptic(α1β2γ2,α2β2γ2,α5β2γ2)and extra-synaptic(α4β2δ)GABAA receptors using the voltage-clamp electrophysiology technique.The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice loss of right reflex(LORR)test.RESULTS Diazepam induced a biphasic potentiation for theα1β2γ2,α2β2γ2 andα5β2γ2 receptor channels,but did not affect theα4β2δreceptor.In contrast to the nanomolar com⁃ponent of potentiation,the second potentiation elicited by micromolar diazepam was insensitive to flumazenil.Midazolam,clonazepam,and loraz⁃epam at 200μmol·L-1 exhibited similar flumaze⁃nil-insensitive effects onα1β2γ2,α2β2γ2 andα5β2γ2 receptors,whereas the potentiation induced by 200μmol·L-1 zolpidem or triazolam was abol⁃ished by flumazenil.Consistent with the in vitro results,flumazenil antagonized the zolpidem(50 mg·kg-1)-induced LORR,but not those induced by 50 mg·kg-1 diazepam or 100 mg·kg-1 midazolam.CONCLUSION The existence of non-classical BZD binding sites on certain GABAA receptor subtypes and the flumazenil-insensitive effects depend on the chemical structures of the allosteric modulators.
