| 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 of?1-AR and?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 of?1-AR and?2-AR antagonists:Antagonists were pretreated,and then DMED were treated to study the mortality of mice or rats.3.Signal pathway of?2B-AR activated by DMED:U2OS-EGFP-NFATc1 cells stably expressed?2B-AR or CHO-PKAcat-EGFP cells stably expressed?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?G?i/o inhibitor?and YM-254890?G?q/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 of?1-AR and?2-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-?2B-AR.Using the NFATc1 nucleus translocation and PKA redistribution,the stably expressed U2OS-EGFP-NFATc1-?2B-AR cells and CHO-PKAcat-EGFP-?2B-AR cells were established.?2-AR agonist DMED,?2-AR antagonist atipamezole hydrochloride?ATI?,?2A-AR antagonist BRL44408,?1/2B-AR antagonist prazosin hydrochloride?prazosin?,?2B-AR antagonist ARC239 and imiloxan were evaluated by NFATc1 nucleus translocation in U2OS-EGFP-NFATc1-?2B-AR cells.DMED,clonidine,guanfacine and?2-AR antagonists idazoxan,ATI were evaluated by PKA redistribution assay in CHO-PKAcat-EGFP-?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-?2B-AR cells and CHO-PKAcat-EGFP-?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 of?1-AR and?2-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 activate?2B-AR.3.1 Establishment of stably-transfected cells.The stably-transfected U2OS-EGFP-NFATc1-?2B-AR cell and CHO-PKAcat-EG FP-?2B-AR cells with significant activity had been established successfully.3.2 NFATc1 translocation after DMED activate?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 activate?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±0.04?M 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 activate?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 activate?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.?1-AR and?2B-AR did not relate to sedative effects of DMED.2.Through?2B-AR and?1-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 G?i/o caused sedative effect of DMED;activation of?2B-AR leading to increase of Ca2+may cause lethal effect of DMED.Innovation:We find the lethal effect of DMED is mediated mostly by?2B-AR and?1-AR,rather than?2A-AR and?2C-AR.The sedative and lethal effect of DMED were due to different?2-AR subtype.The study was used to develop the sedative agents with little side effects. |
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