G αi, and G βγ, subunits have opposite effects on dexmedetomidine-induced sedation

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.

Effect of thienorphine on intestinal transit and isolated guinea-pig ileum contraction

AIM: To evaluate the effect of thienorphine on small intestinal transit in vivo and on guinea-pig ileum (GPI) contraction in vitro . METHODS: The effects of thienorphine on intestinal transit were examined in mice and in isolated GPI. Buprenorphine and morphine served as controls. The distance traveled by the head of the charchol and the total length of the intestine were measured in vivo . Gastrointestinal transit was expressed as a percentage of the distance traveled by the head of the marker relative to the total length of the small intestine. The isolated GPI preparations were connected to an isotonic force transducer and equilibrated for at least 1 h before exposure to drugs. Acetylcholine was used for muscle stimulation. RESULTS: Thienorphine (0.005-1.0 mg/kg, ig ) or bu-prenorphine (0.005-1.0 mg/kg, sc ) dose-dependently significantly inhibited gut transit compared with saline. Thienorphine inhibited gut transit less than buprenorphine. The maximum inhibition by thienorphine on the intestinal transit was 50%-60%, whereas the maximum inhibition by morphine on gut transit was about 100%. Thienorphine also exhibited less inhibition on acetylcholine-induced contraction of GPI, with a maximum inhibition of 65%, compared with 93% inhibition by buprenorphine and 100% inhibition by morphine. Thienorphine induced a concentration-dependent decrease in the basal tonus of spontaneous movement of the GPI, the effect of which was weaker than that with buprenorphine. The duration of the effect of thienorphine on the GPI was longer than that with buprenorphine. CONCLUSION: Thienorphine had less influence, but a longer duration of action on GPI contraction and moderately inhibited intestinal transit.

Mechanism and impact of a novel allosteric AMPA receptor modulator on protection against respiratory depression

OBJECTIVE Respiratory depression hinders the use of anaesthetics and sedative hyp.notics.To explore the mechanism of LCX001 on protection against respiratory depression,a novel AMPA receptor modulator LCX001,synthesized by our Institute of Medicinal Chemistry,is expected to relieve suppressed respiration.METHODS LCX001 was tested to alleviate respiratory depression triggered by opioid(fentanyl and TH-030418),propofol and pentobarbital in the plethysmography recording.The acetic acid writhing and hot-plate tests were conducted to evaluate analgesic effect of LCX001.Binding assay and whole-cell recording were used to analyze the property of LCX001 on positive modulation.The function of AMPA receptors were determined by location of receptors in the membrane and state of channel opening,and both processes were impressed by AMPA receptor regulatory proteins.Ac.cording to the theory,the effect of LCX001 on the expression of stargazin was measured firstly by west.ern blotting.The variation of receptor surface location was observed by live cell imaging.The regula.tion on neuronal Ca^(2+) and cell function was investigated intensively by Ca^(2+) imaging to clarify mecha.nism of LCX001.RESULTS LCX001 effectively rescued and prevented opioid(fentanyl and TH-030418),propofol,and pentobarbital-induced respiratory depression by strengthening respiratory fre.quency and minute ventilation in rats.The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001,in contrast to some ampakines that did not affect analgesia.Fur.thermore,LCX001 potentiated [3 H]AMPA and L-glutamate binding affinity to AMPA receptors,and facili.tated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R).Importantly,appli.cation of LCX001 generated a significant increase in GluA2(R) surface expression in a mechanism of stargazin up-regulation,and restrained opioid-induced abnormal intracellular Ca^(2+) load,which might par.ticipate in breathing modulation.CONCLUSION The novel pharmacological effect and potential new mechanism of LCX001 might promote ampakines to be a therapeutic option for protection against respi.ratory depression.

Wnt1/β-catenin signaling up-regulates spinal VGLUT2 expression to maintain neuropathic pain in mice

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.

Agmatine Prevents Adaptation of the Hippocampal Glutamate System in Chronic Morphine-Treated Rats

Chronic exposure to opioids induces adaptation of glutamate neurotransmission, which plays a crucial role in addiction. Our previous studies revealed that agmatine attenuates opioid addiction and prevents the adaptation of glutamate neurotransmission in the nucleus accumbens of chronic morphine-treated rats. The hippocampus is important for drug addiction; however, whether adaptation of glutamate neurotransmission is modulated by agmatine in the hippocampus remains unknown. Here, we found that continuous pretreatment of rats with ascending doses of morphine for 5 days resulted in an increase in the hip- pocampal extracellular glutamate level induced by nalox- one （2 mg/kg, i.p.） precipitation. Agmatine （20 mg/kg, s.c.） administered concurrently with morphine for 5 days attenuated the elevation of extracellular glutamate levels induced by naloxone precipitation. Furthermore, in the hippocampal synaptosome model, agmatine decreased the release and increased the uptake of glutamate in synapto- somes from chronic morphine-treated rats, which might contribute to the reduced elevation of glutamate levels induced by agrnatine. We also found that expression of the hippocampal NR2B subunit, rather than the NR1 subunit, of N-methyl-D-aspartate receptors （NMDARs） was down-regulated after chronic morphine treatment, and agmatine inhibited this reduction. Taken together, agmatine pre- vented the adaptation of the hippocampal glutamate system caused by chronic exposure to morphine, including mod- ulating extracellular glutamate concentration and NMDAR expression, which might be one of the mechanisms underlying the attenuation of opioid addiction by agmatine.