The Mechanisms Of Dexmedetomidine-induced Sedation And Pharmacodynamics Of Atipamezole

Dexmedetomidine（DMED）is a potent and highly specificα₂-AR agonist which is widely used in veterinary anesthetic and short-term sedation for patients in clinical for its analgesia,sedation neuroprotective and antinociceptive.The application of DMED in clinical becomes higher risk without applicable antagonists ofα₂-AR.Activated-α₂-adrenoceptors couple with pertussitoxin-sensitive G_i/o proteins,which are dissociated with G_α/i and G_βγsubunits.In addition to assosiation with G_αi/o,G_βγsubunits regulated lots of signaling pathways of GPCRs.However,effects of G_βγsubunits on G_α/i/i subunit or the interaction between G_βγsubunits and G_α/i/i subunit has not been illuminated.In present study,we studied on the sedative action ofα₂-AR agonists,the antagonism of ATI against DMED-induced sedation and the mechanism of signaling molecular in regulating DMED-induced sedation.The present study was designed to elucidate the negative regulation of G_βγon DMED-induced sedation by regulating AC-cAMP pathway and phosphorylation of ERK,CREB.ObjectiveThe aims of this study were to evaluate the sedative effects ofα₂-AR agonists and the antagonism of ATI on DMED-induced sedation.Observe the effects of G_αi/o,G_βγsubunits and their downstream signaling molecules on the sedative effect of DMEDContent and Methods1.The sedative effect of DMED and antagonism of ATI on DMED-induced sedation:DMED-induced sedation was evaluated by LORR model,grasping force test,body temperature and blood pressure determination.The antagonism of ATI on sedative effect of DMED was also evaluated in this model.2.The effects of G_αi/o and G_βγsubunits on DMED-induced sedation:Study the effects of modulators or inhibitors of G_αi/oi/o or G_βγsignaling pathway on DMED-induced sedation by establishing LORR model in mice.3.Study on the mechanism of G_αi/o,G_βγsubunits with their downstream signaling molecules on DMED-induced sedation:CHO-α_2A-AR-stable cell line was constructed byα_2A-AR packaging with lentivirus and transfecting into CHO cells.The cAMP content,mRNA and protein levels ofα_2A-AR in CHO-α_2A-AR stably transfected cells were detected to verify receptor function.In CHO-α_2A-AR cells,the effect of the modulators administrated alone or coadministrated with DMED on the cAMP accumulation stimulated by Forskolin（FSK）was detected after administration of activators or inhibitors of downstream signaling molecules of G_βγsubunits.The intracellular calcium ion concentration([Ca²⁺]i)and pERK/pCREB was also detected to demonstrate the key signal molecules involved in DMED-induced sedation.Results1.The sedative effects of and acute toxity ofα₂-AR agonistsDMED dose-dependently(0.10-0.32 mg·kg^-1)inhibited loss of righting reflex（LORR）in mice.The ED₅₀ of DMED was 0.15 mg·kg^-1（i.v.）and 0.18 mg·kg^-1（s.c.）respectively.The immobilization time was prolonged by DMED dose-dependently.Anotherα₂-AR agonist medetomidine（MED）was similar to that of DMED wtih ED₅₀of 0.28 mg·kg^-1（i.v.）and 0.34 mg·kg^-1（s.c.）respectively.The similar results were obtained in rats LORR test.The ED₅₀ of intravaneous injection of DMED and MED were 11.71μg·kg^-1 and 26.58μg·kg^-1.The LD₅₀ value of DMED in mice was 26.73mg·kg^-1（i.v.）2.ATI antagonized DMED-induced sedation and the acute toxity of ATIATI(0.005⁰.050 mg·kg^-1,i.m.)dose dependently antagonized LORR induced by DMED(0.200 mg·kg^-1,i.v.)in mice with ED₅₀ of 12.440μg·kg^-1.ATI(0.050-0.400mg·kg^-1)dose-dependently attenuatedd LORR induced by DMED(0.800 mg·kg^-1,i.v.)with ED₅₀ of 0.170 mg·kg^-1.The ED₅₀ of ATI was 0.062 mg·kg^-1 against DMED(0.200 mg·kg^-1)-induced LORR in rats.The LD₅₀ was 49.470 mg·kg^-1（i.m.）in rats and 47.570 mg·kg^-1（i.m.）in mice.ATI also dose-dependently antagonized DMED-induced hypothermia and muscle relaxation in mice.3.The mechanism of DMED-induced sedation（1）Effects of Gαi_/o-AC-cAMP-PKA signaling molecule on DMED-induced sedationDMED-induced LORR was antagonized significantly after coadministration of cAMP analogue dbcAMP（50 nmol/mouse,i.c.v）or PDE4 inhibitor rolipram（100nmol/mouse,i.c.v）.The ED₅₀ increased to 375.0 nmol/kg and 433.3 nmol/kg respectively.Coadminitration with PKA inhibitor（H89,20 nmol/mouse）had no effect on DMED-induced LORR.The ED₅₀ of DMED was decreased to 163 nmol/kg.DbcAMP,rolipram and H89（i.c.v.）had no effect on mice.（2）Effects of G_βγsignaling molecule on DMED-induced sedationDMED-induced sedative effect was enhanced by G_βγsubunit inhibitor gallein or M119（100 mg/kg,i.p）.The ED₅₀ of DMED decreased to 136.5 nmol/kg and 113.6nmol/kg respectivly.The same results were observed in PLC inhibitor U73122（10nmol/mouse,i.c.v）,PKC inhibitor GF109203X（242 pnmol/mouse,i.c.v）.The ED₅₀of DMED was decreased to 42.5,110.1,and 85.2 nmol/kg respectively.GRK2inhibitor（10 nmol/mouse）also strengthened DMED-induced sedative effect.（3）Mechanisms of G_βγsignal pathway on DMED-induced sedation（1）Effects of G_βγsubunits on intracellular cAMPIn CHO-α_2A-AR cells,βARK1 inhibitor,G_βγsubunit inhibitor gallein,or PKC inhibitor GF109203X（5μM）respectively significantly reduce FSK（10μM）stimulated cAMP accumulation.M119,PLC inhibitor U73122,or MEK inhibitor PD98059 had no effect on the cAMP accumulation induced by FSK.Coadministration with these chemicals had no effect on DMED-induced cAMP inhibition.（2）Effects of G_βγsubunits on[Ca²⁺]iIn CHO-α_2A-AR cells,the intracellular calcium concentration([Ca²⁺]i)was slightly upregulated after DMED,gallein or M119（5μM）administered respectively by calcium imaging experiments.Coadministrated G_βγsubunits inhibitor with DMED significantly increased[Ca²⁺]i in cytoplasm.（3）Effects of DMED and downstream molecule of G_βγsubunits on pERKpERK was significantly activated after gallein（5μM）administrated for 5-30 min in CHO-α_2A-AR cells.Coadministrated with gallein,the upregulation of pERK was potentiated under DMED（5μM）treatment with extended time.The effect of M119 is similar to that of gallein.PKC inhibitor GF109203X（5μM）significantly increased DMED-induced upregulation of pERK at 5 and 15 min after coadministration with DMED（5μM）.βARK1 inhibitor（5μM）significantly enhanced DMED-induced up-regulation of pERK at 5-30 min after coadministration with DMED（5μM）.This result suggests that the activation of pERK is closely related to the sedative-hypnotic effect of DMED.（4）Effects of DMED and downstream molecule of G_βγsubunits on pCREBIn CHO-α_2A-AR cell,pCREB increased significantly at 5 min,peaked at 15 min,and returned to control at 60min after DMED（5μM）administration.pCREB was significantly up-regulated after gallein（5μM）administrated for 5-15 min in CHO-α_2A-AR cells.Coadministrated with gallein,the upregulation of pCREB was potentiated under DMED（5μM）treatment with extended time for 30 min.The effect of M119 is similar to that of gallein.The PKC inhibitor GF109203X（5μM）significantly increased DMED-induced upregulation of pERK at 5 and 15 min after coadministration with DMED（5μM）.βARK1 inhibitor（5μM）significantly enhanced DMED-induced up-regulation of pCREB at 5-30 min after coadministration with DMED（5μM）.（4）Mechanisms of G_αi/o-cAMP-PKA signal pathway on DMED-induced sedationInα_2A-AR-CHO cells,pERK was significantly strengthened after DMED（5μM）administration.This effect was inhibited at 15 min after coadministrated dbcAMP（5μM）with DMED（5μM）.Coadministrated rolipram（5μM）with DMED also inhibited the up-regulation of pERK.After DMED（5μM）was coadministered with dbcAMP（5μM）,dbcAMP had no effect on the up-regulation of pCREB levels induced by DMED.Similar results were observed after coadministration of DMED（5μM）and rolipram（5μM）.DbcAMP or rolipram alone had no effect on pERK/pCREB.Conclusion1.DMED has strong sedative and hypnotic effect,including reducing body temperature,blood pressure,muscle tension and spontaneous activity which can be antagonized by ATI.ATI is a safety and effectiveα₂-AR antagonist which can be developed for clinical application ofα₂-AR antagonists.2.G_αi/o-AC-cAMP-PKA,G_βγ–PLC-PKC and GRKs signaling pathway play an important role in regulating DMED-induced sedation.G_βγ–PLC-PKC and GRKs signaling pathway have negative regulation on DMED-induced sedation.3.Inhibition of G_βγsubunits and their downstream signaling molecules enhanced the sedative effect of DMED.G_βγsubunits with their signal molecules may have a negative regulatory effect on receptor function afterα₂-AR activation.pERK is closely related to DMED-induced sedation.pCREB may be as a downstream indicator of DMED mediating sedation.