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 explore the mechanism of G_(αi) and G_(βγ) subunits on dexmedetomidine(DMED)-induced sedation.METHODS Kunming mice were randomly placed into three groups(DMED group,DMED+dbcAMP/rolipram/gallein/M119 gr...OBJECTIVE To explore the mechanism of G_(αi) and G_(βγ) subunits on dexmedetomidine(DMED)-induced sedation.METHODS Kunming mice were randomly placed into three groups(DMED group,DMED+dbcAMP/rolipram/gallein/M119 group,dbcAMP/rolipram/gallein/M119 group) to explore the regulation of dbcAMP/rolipram/gallein/M119 on DMED-induced sedation by establishing loss of righting reflex(LORR) model.DbcAMP/rolipram was intracerebroventricular injected and gallein/M119 was intraperitoneal injected 15 min before DMED intravenous injection.In CHO-α2 A-AR cells,after administration of DMED/gallein/M119,the regulation on the cAMP accumulation stimulated by Forskolin(FSK) was detected,so was the intracellular calcium ion concentration([Ca2 + ]i.The levels of pERK/pCREB were detected by Western Blot to explore the key signal molecules involved in DMED-induced sedation.RESULTS The ED50 of DMED-induced LORR(200.0 nmol·kg^(-1)) was increased to 375.0 or433.3 nmol·kg^(-1) by pre-treatment with cAMP analog dbcAMP(50 nmol/5μl per mouse) or phosphodies.terase 4 inhibitor rolipram(100 nmol/5μl per mouse).In addition,the ED50 of DMED-induced LORR was decreased to 113.6 or 136.5 nmol·kg^(-1) when pre-treated with G_(βγ) subunits inhibitor M119(100 mg·kg^(-1))or gallein(100 mg·kg^(-1)) respectively.Administration of dbcAMP,rolipram,gallein or M119 alone had little effect on LORR of mice.Gallein(10 μmol·L^(-1)) significantly inhibited forskolin-stimulated cAMP accumu.lation in CHO-α2A-AR cells.Compared with G_(βγ) subunits inhibitors or DMED alone,[Ca^(2+)]i and pERK1/2 significantly increased after co-administration of G_(βγ) subunits inhibitors with DMED.DbcAMP(5 μmol·L^(-1))or rolipram(5 μmol·L^(-1)) alone had little effect on ERK1/2 phosphorylation,but decreased DMEDinduced ERK1/2 phosphorylation after co-administration with DMED.G_(βγ) subunit inhibitors treatment increased DMED-induced phosphorylation of CREB,whereas dbcAMP or rolipram had little effect on pCREB induced by DMED.CONCLUSION G_(βγ) subunits might inhibit DMED-induced sedation through cAMP and pERK1/2 pathway,which was opposite to G_(αi) subuint.展开更多
OBJECTIVE Thienorphine,a new oripavine derivative,has shown to possess stronger antinociceptive effects and better oral bioavailability compared to buprenorphine.The present study examines the effect of thienorphine o...OBJECTIVE Thienorphine,a new oripavine derivative,has shown to possess stronger antinociceptive effects and better oral bioavailability compared to buprenorphine.The present study examines the effect of thienorphine on c AMP-dependent protein kinase A(PKA) activity in CHO cells expressing μ-,κ-,δ-and ORL1 receptors.In addition,we further examined its analgesic effect in vivo.METHODS The effect of thienorphine on cA MP-dependent PKA redistribution and cA MP inhibition were analyzed in CHO-PKAcatEGFP cells.PKA redistribution assays in CHO-PKAcatEGFP cells stably expressing μ-,κ-,δ-and ORL1 receptors were analyzed by high-throughput screening system to elucidate the efficacy of agonists or antagonists on opioid receptors.Moroever,the antinociceptive effects of thienorphine in vivo were examined using hot plate test.RESULTS Briefly,the maximum inhibition of thienorphine on PKA activity was about 36%,100%,100%and 12% in CHO-μ/κ/δ/ORL1-PKAcatE GFP cel s,respectively.In addition,thienorphine concentrationdependently inhibited the PKA activity with EC50 value of(22.7±18.1) nmol·L^(-1) in CHO-κ-PKAcatE GFP cels and(12.4±7.7) nmol·L^(-1) in CHO-δ-PKAcatE GFP cells.Thienorphine induced approximately 50%antinociceptive effect in mice lacking μ receptors compared to their wild-type controls(P<0.05).Also,the κ and δ selective antagonist nor-binaltorphimine,naltrindole decreased approximately 50%-60% in % MPE of theinorphine in μ-KO mice,respectively.The ORL1 receptor selective antagonist J113397 had no effect in %MPE of theinorphine in μ-KO mice.CONCLUSION Thienorphine induces analgesia through bindingκ-and δ-,or by partially binding μ-opioid receptor,thus further regulating the cAMP-PKA activity.Therefore,thienorphine may be used in acute or chronic pain with minimal addictive potential.展开更多
OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs ...OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs in mouse models of depression,the reason for these strain differences remains unclear.Here,therefore,in the present study,we examined immobility time and locomotor activity in two mouse strains,namely,C57BL/6 J and DBA/2 J mice,and the effects of the SSRIs fluoxetine.Furthermore,we analyzed 5-HT transporter binding and reuptake inhibition in both strains to explore their relationship with the immobility and locomotor activity effects of the three SSRIs in these two mouse strains.METHODS Strain differences in SSRI effects in the tail suspension test(TST) and forced swimming test(FST).To initiate our studies,we sought to confirm that SERT strain variation did not alter SERT protein expression,5-HT recognition,or uptake activity when expressed in C57BL/6 J and DBA/2 J mice.Radioligand binding assays were conducted to determine the affinity of the SSRIs for the 5-HT transporters in the two mouse strains.RESULTS SSRI citalopram dose-dependently reduced immobility time in both the FST and TST in DBA/2 J but not C57BL/6 J mouse strains,whereas fluoxetine showed opposite results.Paroxetine reduced immobility time similarly in both strains.The affinity of citalopram for the 5-HT transporter in DBA/2 J mice was 700-fold higher than that for in C57BL/6 J mice,whereas the affinity of fluoxetine in C57BL/6 J mice was 100-fold higher than that in the DBA/2 J mouse.Furthermore,High citalopram concentrations were required to [3 H]5-HT uptake in C57BL/6 J but not DBA/2 J mouse cortical synaptosomes,whereas fluoxetine also showed opposite results.CONCLUSION Immobility duration depends on 5-HT transporter binding levels,leading to apparent strain differences in immobility time in FST and TST.Furthermore,differences in 5-HT transporter binding may cause variations in SSRI responses on behaviors.SERT mutation mice maintained sensitivity to paroxetine,an antidepressant that is unaffected by the mouse mutation.Therefore,the background strain of these mice likely contributes to the acute behavioral actions of SSRIs in immobility time.These differences may help to explain some of the discrepancies in studies that used these strains of mice to examine the role of 5-HT in mouse models of depression.Future studies should investigate additional neural substrates and molecular mechanisms underlying strain variations in mouse models of depression to help identify genetic predispositions to this disorder in humans.展开更多
OBJECTIVE The present study was aimed to investigate the role of Wnt/β-catenin sig.naling in spinal VGLUT2 regulation and neuropathic pain.METHODS To elucidate the association be.tween VGLUT2 and neuropathic pain,we ...OBJECTIVE The present study was aimed to investigate the role of Wnt/β-catenin sig.naling in spinal VGLUT2 regulation and neuropathic pain.METHODS To elucidate the association be.tween VGLUT2 and neuropathic pain,we determined the expression and distribution characteristics of VGLUT2 in mice subjected to spared nerve injury(SNI),and then observed the effects of two VGLUT2 targeting shRNAs on mechanical allodynia and glutamate release.The effects of Wnt/β-catenin signal.ing on VGLUT2 expression and pain behavior were investigated by using Wnt agonist,Wnt1,and Wnt/β-catenin pathway inhibitor XAV939 in SNI mice.RESULTS SNI surgery induced significant up-regula.tion of VGLUT2 on postoperative days 7,14,and 21.Double immunofluorescence labeling of VGLUT2 with NeuN,MAP2,Iba-1,or GFAP showed that VGLUT2 was mainly expressed in neurons in the dor.sal horn of the spinal cord after SNI(NeuN,MAP2).Intrathecal administration of VGLUT2 shRNAs be.fore or after SNI surgery significantly decreased mechanical allodynia and glutamate release.Mean.while,Wnt1/β-catenin signaling increased significantly after SNI surgery.Over-expression of β-catenin in PC12 cells increased VGLUT2 protein level,intrathecal administration of Wnt agonist or Wnt1 signifi.cantly increased VGLUT2 protein expression in spinal cord,while Wnt/β-catenin pathway inhibitor XAV939 decreased VGLUT2 expression in PC12 cells and spinal cord.Additionally,intrathecal admin.istration of XAV939 7 days after SNI significantly attenuated mechanical allodynia in mice,which was in accordance with down-regulation of VGLUT2 protein levels.VGLUT2 shRNAs significantly attenuat.ed Wnt agonist or Wnt1 induced mechanical allodynia.CONCLUSION Wnt1/β-catenin signaling path.way up-regu-lates the spinal VGLUT2 expression,and this regulation is involved in neuropathic pain behavior.展开更多
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.
文摘OBJECTIVE To explore the mechanism of G_(αi) and G_(βγ) subunits on dexmedetomidine(DMED)-induced sedation.METHODS Kunming mice were randomly placed into three groups(DMED group,DMED+dbcAMP/rolipram/gallein/M119 group,dbcAMP/rolipram/gallein/M119 group) to explore the regulation of dbcAMP/rolipram/gallein/M119 on DMED-induced sedation by establishing loss of righting reflex(LORR) model.DbcAMP/rolipram was intracerebroventricular injected and gallein/M119 was intraperitoneal injected 15 min before DMED intravenous injection.In CHO-α2 A-AR cells,after administration of DMED/gallein/M119,the regulation on the cAMP accumulation stimulated by Forskolin(FSK) was detected,so was the intracellular calcium ion concentration([Ca2 + ]i.The levels of pERK/pCREB were detected by Western Blot to explore the key signal molecules involved in DMED-induced sedation.RESULTS The ED50 of DMED-induced LORR(200.0 nmol·kg^(-1)) was increased to 375.0 or433.3 nmol·kg^(-1) by pre-treatment with cAMP analog dbcAMP(50 nmol/5μl per mouse) or phosphodies.terase 4 inhibitor rolipram(100 nmol/5μl per mouse).In addition,the ED50 of DMED-induced LORR was decreased to 113.6 or 136.5 nmol·kg^(-1) when pre-treated with G_(βγ) subunits inhibitor M119(100 mg·kg^(-1))or gallein(100 mg·kg^(-1)) respectively.Administration of dbcAMP,rolipram,gallein or M119 alone had little effect on LORR of mice.Gallein(10 μmol·L^(-1)) significantly inhibited forskolin-stimulated cAMP accumu.lation in CHO-α2A-AR cells.Compared with G_(βγ) subunits inhibitors or DMED alone,[Ca^(2+)]i and pERK1/2 significantly increased after co-administration of G_(βγ) subunits inhibitors with DMED.DbcAMP(5 μmol·L^(-1))or rolipram(5 μmol·L^(-1)) alone had little effect on ERK1/2 phosphorylation,but decreased DMEDinduced ERK1/2 phosphorylation after co-administration with DMED.G_(βγ) subunit inhibitors treatment increased DMED-induced phosphorylation of CREB,whereas dbcAMP or rolipram had little effect on pCREB induced by DMED.CONCLUSION G_(βγ) subunits might inhibit DMED-induced sedation through cAMP and pERK1/2 pathway,which was opposite to G_(αi) subuint.
基金National Natural Science Foundation of China(8147319481773709).
文摘OBJECTIVE Thienorphine,a new oripavine derivative,has shown to possess stronger antinociceptive effects and better oral bioavailability compared to buprenorphine.The present study examines the effect of thienorphine on c AMP-dependent protein kinase A(PKA) activity in CHO cells expressing μ-,κ-,δ-and ORL1 receptors.In addition,we further examined its analgesic effect in vivo.METHODS The effect of thienorphine on cA MP-dependent PKA redistribution and cA MP inhibition were analyzed in CHO-PKAcatEGFP cells.PKA redistribution assays in CHO-PKAcatEGFP cells stably expressing μ-,κ-,δ-and ORL1 receptors were analyzed by high-throughput screening system to elucidate the efficacy of agonists or antagonists on opioid receptors.Moroever,the antinociceptive effects of thienorphine in vivo were examined using hot plate test.RESULTS Briefly,the maximum inhibition of thienorphine on PKA activity was about 36%,100%,100%and 12% in CHO-μ/κ/δ/ORL1-PKAcatE GFP cel s,respectively.In addition,thienorphine concentrationdependently inhibited the PKA activity with EC50 value of(22.7±18.1) nmol·L^(-1) in CHO-κ-PKAcatE GFP cels and(12.4±7.7) nmol·L^(-1) in CHO-δ-PKAcatE GFP cells.Thienorphine induced approximately 50%antinociceptive effect in mice lacking μ receptors compared to their wild-type controls(P<0.05).Also,the κ and δ selective antagonist nor-binaltorphimine,naltrindole decreased approximately 50%-60% in % MPE of theinorphine in μ-KO mice,respectively.The ORL1 receptor selective antagonist J113397 had no effect in %MPE of theinorphine in μ-KO mice.CONCLUSION Thienorphine induces analgesia through bindingκ-and δ-,or by partially binding μ-opioid receptor,thus further regulating the cAMP-PKA activity.Therefore,thienorphine may be used in acute or chronic pain with minimal addictive potential.
文摘OBJECTIVE Selective serotonin reuptake inhibitors(SSRIs) bind 5-HT transporters,leading to the accumulation of 5-HT and amelioration of depression.Although different mouse strain showed different sensitivity to SSRIs in mouse models of depression,the reason for these strain differences remains unclear.Here,therefore,in the present study,we examined immobility time and locomotor activity in two mouse strains,namely,C57BL/6 J and DBA/2 J mice,and the effects of the SSRIs fluoxetine.Furthermore,we analyzed 5-HT transporter binding and reuptake inhibition in both strains to explore their relationship with the immobility and locomotor activity effects of the three SSRIs in these two mouse strains.METHODS Strain differences in SSRI effects in the tail suspension test(TST) and forced swimming test(FST).To initiate our studies,we sought to confirm that SERT strain variation did not alter SERT protein expression,5-HT recognition,or uptake activity when expressed in C57BL/6 J and DBA/2 J mice.Radioligand binding assays were conducted to determine the affinity of the SSRIs for the 5-HT transporters in the two mouse strains.RESULTS SSRI citalopram dose-dependently reduced immobility time in both the FST and TST in DBA/2 J but not C57BL/6 J mouse strains,whereas fluoxetine showed opposite results.Paroxetine reduced immobility time similarly in both strains.The affinity of citalopram for the 5-HT transporter in DBA/2 J mice was 700-fold higher than that for in C57BL/6 J mice,whereas the affinity of fluoxetine in C57BL/6 J mice was 100-fold higher than that in the DBA/2 J mouse.Furthermore,High citalopram concentrations were required to [3 H]5-HT uptake in C57BL/6 J but not DBA/2 J mouse cortical synaptosomes,whereas fluoxetine also showed opposite results.CONCLUSION Immobility duration depends on 5-HT transporter binding levels,leading to apparent strain differences in immobility time in FST and TST.Furthermore,differences in 5-HT transporter binding may cause variations in SSRI responses on behaviors.SERT mutation mice maintained sensitivity to paroxetine,an antidepressant that is unaffected by the mouse mutation.Therefore,the background strain of these mice likely contributes to the acute behavioral actions of SSRIs in immobility time.These differences may help to explain some of the discrepancies in studies that used these strains of mice to examine the role of 5-HT in mouse models of depression.Future studies should investigate additional neural substrates and molecular mechanisms underlying strain variations in mouse models of depression to help identify genetic predispositions to this disorder in humans.
基金supported by National Natural Science Foundation of China(81200850) Beijing Natural Science Foundation(7123224) National Science and Technology Major Project of China(2012ZX09301003-001)
文摘OBJECTIVE The present study was aimed to investigate the role of Wnt/β-catenin sig.naling in spinal VGLUT2 regulation and neuropathic pain.METHODS To elucidate the association be.tween VGLUT2 and neuropathic pain,we determined the expression and distribution characteristics of VGLUT2 in mice subjected to spared nerve injury(SNI),and then observed the effects of two VGLUT2 targeting shRNAs on mechanical allodynia and glutamate release.The effects of Wnt/β-catenin signal.ing on VGLUT2 expression and pain behavior were investigated by using Wnt agonist,Wnt1,and Wnt/β-catenin pathway inhibitor XAV939 in SNI mice.RESULTS SNI surgery induced significant up-regula.tion of VGLUT2 on postoperative days 7,14,and 21.Double immunofluorescence labeling of VGLUT2 with NeuN,MAP2,Iba-1,or GFAP showed that VGLUT2 was mainly expressed in neurons in the dor.sal horn of the spinal cord after SNI(NeuN,MAP2).Intrathecal administration of VGLUT2 shRNAs be.fore or after SNI surgery significantly decreased mechanical allodynia and glutamate release.Mean.while,Wnt1/β-catenin signaling increased significantly after SNI surgery.Over-expression of β-catenin in PC12 cells increased VGLUT2 protein level,intrathecal administration of Wnt agonist or Wnt1 signifi.cantly increased VGLUT2 protein expression in spinal cord,while Wnt/β-catenin pathway inhibitor XAV939 decreased VGLUT2 expression in PC12 cells and spinal cord.Additionally,intrathecal admin.istration of XAV939 7 days after SNI significantly attenuated mechanical allodynia in mice,which was in accordance with down-regulation of VGLUT2 protein levels.VGLUT2 shRNAs significantly attenuat.ed Wnt agonist or Wnt1 induced mechanical allodynia.CONCLUSION Wnt1/β-catenin signaling path.way up-regu-lates the spinal VGLUT2 expression,and this regulation is involved in neuropathic pain behavior.
