Mechanism Of The Toxicity And Sedative Effects Induced By Dexmedetomidine Hydrochloride On Receptor Subtype

Objective:To study the receptor types and post-receptor molecular pathways that included in dexmetomidine hydrochloride（DMED）induced toxicity.And then demonstrate the different mechanisms between toxicity and sedation induced by DMED.Content:1.Anti-sedative effects ofα₁-AR andα_2B-AR antagonists:Models of mice lossing righting reflex induced by DMED were used to identify anti-sedative effects of different antagonists.2.Anti-lethal effects ofα₁-AR andα₂-AR antagonists:Antagonists were pretreated,and then DMED were treated to study the mortality of mice or rats.3.Signal pathway ofα_2B-AR activated by DMED:U2OS-EGFP-NFATc1 cells stably expressedα_2B-AR or CHO-PKAcat-EGFP cells stably expressedα_2B-AR were established.NFATc1 nucleus translocation or redistribution of PKA was analysed for activation of signal pathway related to NFATc1 or AC-cAMP-PKA signal pathway by different agents.4.Mechanism of toxic and sedative effects for DMED:The toxicity and sedation induced by DMED were studied by the pretreatment of PTX（Gαi/o inhibitor）and YM-254890（Gαq/11 inhibitor）.The toxicity induced by DMED was also studied by the pretreatment of U73122（inhibitor of phospholipase C）,BAPTA(chelator for Ca²⁺),verapamil and nimodipine.Methods:1.Prazosin,imiloxan and HEAT were pretreated 15 min before DMED（0.17 mg/kg,i.v）treatment in mice.The anti-sedative effects were evaluated by rate of loss of righting reflex（LORR）,induction time and immobilization time of LORR.2.Mice or rats were treated with DMED as control.Antagonists ofα₁-AR andα₂-AR were pretreated 15 min before DMED treatment,the mortality rate was recorded for 7days.The pulmonary pathological slides of mice by hematoxylin-eosin staining were made to study toxic effects of DMED.3.U2OS cells stably expressed the EGFP-NFATc1 or CHO cells stably expressed PKAcat-EGFP was transfected with recombinant plasmid pcDNA3.1-Hygro-α_2B-AR.Using the NFATc1 nucleus translocation and PKA redistribution,the stably expressed U2OS-EGFP-NFATc1-α_2B-AR cells and CHO-PKAcat-EGFP-α_2B-AR cells were established.α₂-AR agonist DMED,α₂-AR antagonist atipamezole hydrochloride（ATI）,α_2A-AR antagonist BRL44408,α_1/2B-AR antagonist prazosin hydrochloride（prazosin）,α_2B-AR antagonist ARC239 and imiloxan were evaluated by NFATc1 nucleus translocation in U2OS-EGFP-NFATc1-α_2B-AR cells.DMED,clonidine,guanfacine andα₂-AR antagonists idazoxan,ATI were evaluated by PKA redistribution assay in CHO-PKAcat-EGFP-α_2B-AR cells.4.YM-254890 were pretreated 30 min before DMED（i.v）treatment,PTX were pretreated 7 days before DMED（i.v）treatment,in order to study the toxic or sedative effects in mice;In vitro,YM-254890 or PTX were co-administrated w ith DMED to evaluate the NFATc1 translocation and PKA redistribution in U2OS-EGFP-NFATc1-α_2B-AR cells and CHO-PKAcat-EGFP-α_2B-AR cells respectively.U73122,BAPTA,verapamil and nimodipine were pretreated 15 min before DMED（i.v）to study the toxic effects in mice.Results:1.Prazosin,imiloxan and HEAT had no effect on the LORR induced by DMED in mice.2.Anti-lethal effects ofα₁-AR andα₂-AR antagonists.2.1 Effects of different antagonists on toxicity induced by DMED in mice.ATI had no anti-lethal effect.ARC239 pretreatment significantly decreased the mortality rate induced by DMED（25.6 mg/kg,i.v.）on the first day（P<0.001）and in 7days（P<0.05）.Prazosin,ARC239,HEAT,BRL44408 and JP1302,significantly decreased the mortality induced by DMED on the first day,but has no effect on mortality rate in 7 days.2.2 Effects of different antagonists on toxicity induced by DMED in rats.Prazosin,HEAT and ARC239 decreased the mortality rate induced by DMED（8mg/kg,i.p）on the first day and in 7 days.ATI had anti-lethal effects in 7 days.Prazosin significantly decreased mortality rate induced by DMED（8 mg/kg,i.v）on the first day and in 7 days,but ATI had no effect.Prazosin decreased mortality rate induced by DMED（8,16,24 mg/kg,i.v,0.02 mL/min）on the first day and in 7 days.ATI significantly decreased the mortality rate on the first day,but had no effect on mortality rate in 7 days.2.3 Pulmonary injure of mice treated with DMEDDMED（25.6 mg/kg,i.v）caused the mice death with acute pulmonary congestion.HE staining of mice lung tissue revealed obvious pulmonary vascular congestion,alveolar rupture and influx of red blood cells.3.Signal pathway after DMED activateα_2B-AR.3.1 Establishment of stably-transfected cells.The stably-transfected U2OS-EGFP-NFATc1-α_2B-AR cell and CHO-PKAcat-EG FP-α_2B-AR cells with significant activity had been established successfully.3.2 NFATc1 translocation after DMED activateα_2B-ARDMED concentration-dependently induced nucleus translocation of NFATc1 wit h EC₅₀:（2.62±0.07）nM.ATI,ARC239,prazosin,imiloxan,BRL44408 concentr ation-dependently decreased the effect of DMED（10^-77 M）,with IC₅₀0 89.05±0.13,11.13±33.53,493.40±0.25,103.50±2.35,931±0.21 nM respectively.The pharmacodyna mic effect of different antagonists was ARC239?ATI?imiloxan?prazosin?B RL44408.3.3 PKA redistribution after DMED activateα_2B-AR.DMED,clonidine,and guanfacine could activate AC-cAMP-PKA signal pathwa y and concentration-dependently increased the PKAcat-EGFP aggregates with EC₅₀:1.15±0.11,11.00±0.44,58.50±0.08 nM respectively.ATI and Imidazoleconcentration-dependently decreased the effect of DMED(10^-7M)with IC₅₀:565.60±0.21 nM and 5.53±0.04μM respectively.4.Mechanism of toxic and sedative effects for DMED.4.1 Effects of YM-254890 on toxicity and sedation induced by DMED:YM-254890 had no anti-lethal effect and anti-sedative after treatment with DMED in mi ce.4.2 Effects of PTX on toxicity and sedative effect of DMED.PTX had no anti-lethal effects,but had anti-sedative effect induced by DME D in mice.4.3 Effect of YM-254890 when DMED activateα_2B-AR.YM-254890 could concentration-dependently decreased the effect of DMED(10^-7M),and IC50 was（103.50±2.34）nM.4.4 Effect of PTX when DMED activateα_2B-AR.PTX has no effect on NFATc1 translocation induced by DMED.4.5 Effects of U73122,BAPTA,verapamil and nimodipine on toxicity effect o f DMED.U73122 had no anti-lethal effect.BAPTA,verapamil and nimodipine had signi ficant anti-lethal effect.Conclusion:1.α₁-AR andα_2B-AR did not relate to sedative effects of DMED.2.Throughα_2B-AR andα₁-AR,high-dose of DMED caused death of mice and rats,the pulmonary congestion might related to lethal effect of DMED.3.Activation of signal pathway by Gαi/o caused sedative effect of DMED;activation ofα_2B-AR leading to increase of Ca²⁺may cause lethal effect of DMED.Innovation:We find the lethal effect of DMED is mediated mostly byα_2B-AR andα₁-AR,rather thanα_2A-AR andα_2C-AR.The sedative and lethal effect of DMED were due to differentα₂-AR subtype.The study was used to develop the sedative agents with little side effects.