Possible Mechanisms Of Fentanyl-induced Respiratory Depression

Until now,opioid drugs remain the first choice for the treatment of moderate to severe pain in clinical.Among them,fentanyl compounds have good analgesic activity,various dosage forms and rapid onset of action.They have become the most widely used intraoperative analgesic drugs in the world.Fentanyl compounds can also be used as an anesthetic auxiliary drugs in combination with anesthetics.However,due to certain side-effects of the central nervous system,such as addiction,tolerance,and especially respiratory depression,this type of compound greatly limits the clinical application and becomes the main reason for patients to stop taking drugs.Although clinical use of opioid receptor antagonists,such as naloxone,can reverse opioid-induced respiratory depression,these drugs also can significantly reduce the analgesic effect of fentanyl drugs.Therefore,through the study of the basic research of fentanyl-induced respiratory depression,the development of new analgesic drugs with exact analgesic effect and low side effects of respiratory depression has become an important research area.Fentanyl compounds plays a biological role in the body by stimulating opioid receptors.Opioid receptors primarily transduce signals into the cell after receptor activation by binding to extracellular specific ligands（such as fentanyl）on the surface of the cell membrane.On the one hand,the G protein-coupled receptor signaling system exerts a biological effect:after the ligand binds the receptor,the conformational change of the G protein coupled to the receptor directly leads to the separation of the G_αand G_βγsubunits.Activated subunits transmit and amplify signals by acting on different effector molecules,mediating multiple signaling pathways in the cell,including activation of G protein-coupled receptor kinases（GRKs）and protein kinase C（PKC）;inhibits the activity of adenylate cyclase（AC）.On the other hand,opioid receptors can also generate signals through non-G protein signaling pathways,such as theβ-arrestin pathway:β-arrestin is a negative feedback regulatory protein of G protein signaling,involved in receptor desensitization,internalization and recycling,and termination G protein signal transduction.In addition,β-arrestin can also be used as a scaffold protein to recruit different signaling molecules,mediating G protein-independent signaling pathways.Epac（Exchange protein directly activated by cAMP）is also a downstream target molecule of cAMP,which can interact with PKA（protein kinase A）or mediate biological effects alone.Studies have confirmed thatβ-arrestin 2 knockout mice（βarr2-KO）show enhanced morphine analgesia,no significant tolerance to chronic morphine treatment and a significant reduction in respiratory depression,suggesting thatβ-arrestin 2 may regulate morphine-induced respiratory depression.Whether the fentanyl compounds-induced respiratory depression is related toβ-arrestin 2 is not clear;other studies have shown that PDE4（phosphodiesterase 4）inhibitor can also reverse the morphine-induced respiratory depression by increasing intracellular cAMP content of neurons.But until now the molecular mechanism of cAMP in fentanyl-induced respiratory depression remains inconclusive.There is a clear differences between downstream of signal transduction in different opioid agonists after stimulating opioid receptors,such as morphine and fentanyl.Therefore,the biological mechanisms of respiratory depression induced by them may be different too.To explain these problems,we explored the opioid receptor signaling pathway and other related receptors that may affect the pathway of opioid receptors.First,we verified whetherβ-arrestin 2 participates in the fentanyl-induced respiratory depression effect.It is planned to use primary cells,normal and transgenic whole animals to observe the relationship between fentanyl-induced respiratory depression andβ-arrestin 2;The role of the G protein signaling pathway in fentanyl-induced respiratory depression will be analyzed on cAMP levels and cAMP downstream PKA,Epac levels;We also combined with other receptors,such as:adrenergic receptors,NMDA receptors,attempt to elucidate the fentanyl induced respiratory depression.Objective:The aim of this study is to verify that the fentanyl-induced respiratory depression is related toβ-arrestin 2 or G protein signaling pathway,and further explore the specific molecules that are related to the respiratory depression,and to investigate the possible mechanisms of fentanyl compounds-induced respiratory depression.Methods:1.The correlation betweenβ-arrestin 2 signaling pathway and fentanyl-induced respiratory depressionPrimary neuron culture was used to study the effect on the expression ofβ-arrestin 2after fentanyl compounds i.v.administration.The expression ofβ-arrestin 2 in rat cortex,hippocampus and medulla oblongata were detected at 30 and 120 min after acute fentanyl-001 administration.Administration of theβ-arrestin 2 pathway inhibitor in normal animal model to study the association ofβ-arrestin 2 with the respiratory depression effect.Finally,further confirmation was gained on theβ-arrestin 2 knockout mice.2.The role of G protein signaling pathway in fentanyl-induced respiratory depression Using GIRK（G protein gated inwardly rectifying K channels）inhibitor to investigate the effect of G_βγdownstream signaling on fentanyl-induced respiratory depression in rats;i.c.v.administration of cAMP analogue 8-Br-cAMP or i.v.administration of PDE4inhibitor Rolipram to investigate the effect of cAMP on fentanyl-induced respiratory depression;i.c.v.administration of PKA（protein kinase A）inhibitors H89,KT5720,and Rp-cAMPs to examine the effects of PKA on fentanyl-induced respiratory depression;to investigate the relationship between adrenergic receptor,NMDA receptor and fentanyl-induced respiratory depression in rats.3.Epac（exchange protein directly activated by cAMP）signaling pathway and its effect on respiratory depression induced by fentanylUsing i.c.v.administration of Epac agonist 8-pCPT to investigate the effect of Epac in fentanyl-induced respiratory depression;the effect of MEK inhibitors U0126 and SL327 in fentanyl and morphine induced respiratory depression were investigated by i.c.v.administration.The effect of 8-pCPT on fentanyl and morphine-induced ERK phosphorylation on CHO-μcell line were examined.The distribution of intracellular phosphorylated ERK was examined after fentanyl and morphine administration on PC12 cell line.Results:1.β-arrestin 2 does not participate in fentanyl-induced respiratory depressionAcute treatment with morphine（1μM）and fentanyl-001（1,10μM）for 1 h did not affect the expression ofβ-arrestin 2 in cortical primary neurons;acute treatment with fentanyl analog（20μg/kg,i.v.）after 30 min,the expression ofβ-arrestin 2 in the medulla and hippocampus was unchanged（P>0.05）,and the expression ofβ-arrestin 2in the cortex was decreased（P<0.05）.Fentanyl-001（20μg/kg,i.v.）after 120 min of acute treatment,the expression ofβ-arrestin 2 in the medulla,hippocampus and cortex of the rats was unchanged（P>0.05）.Administration of GRK2 inhibitor（25μg/site in rats,50μg/site in mice）did not affect fentanyl-induced respiratory depression（P>0.05）;administration of morphine（50 mg/kg,s.c.）and fentanyl（0.4 mg/kg,s.c.）inβarr2-KO mice showed that after 40-90 min morphine administration,the increase in the inspiratory time(F_2,162=9.53,P<0.01)and the expiratory time(F_2,162=7.59,P<0.01)caused by morphine was significantly decreased compared to WT mice.There was no significant difference in respiratory rate,inspiratory time,expiratory time and minute ventilation betweenβarr2-KO animals and WT animals after 10 minutes of fentanyl administration（P>0.05）.2.The role of G protein signaling pathway in fentanyl-induced respiratory depression GIRK inhibitor Tertiapin-Q（0.5,2.0μg/site,i.c.v.）dose-dependently relieved respiratory rate depresssion due to fentanyl administration(F_2,114=5.87,P<0.05),and increased inspiratory time(F_2,108=4.97,P<0.05),there was an increasing trend of expiratory time but no statistical difference（P>0.05）.There was no significant difference between the minute ventilation caused by fentanyl（P>0.05）in these groups.The cAMP analog 8-Br-cAMP（100μg/site,i.c.v.）did not affect the decrease in respiratory rate,inspiratory time,and expiratory time due to administration of fentanyl（P>0.05）.The PDE inhibitor rolipram（1,2 mg/kg,i.v.）did not affect the respiratory rate,inspiratory time,and expiratory time due to fentanyl administration（P>0.05）.PKA inhibitor H89（50μg/site,i.c.v.）significantly increased the respiratory rate caused by fentanyl administration(F_2,108=17.94,P<0.001),and reduced inspiratory time(F_2,108=6.48,P<0.01)and expiratory time(F_2,108=12.30,P<0.001),but did not affect the decrease in minute ventilation caused by fentanyl（P>0.05）.PKA inhibitor KT5720（10μg/site,i.c.v.）significantly increased the respiratory rate(F_1,48=26.93,P<0.001),and the inspiratory time(F_1,48=9.60,P<0.05)and expiratory time(F_1,48=20.67,P<0.01)was decreased.Rp-cAMPs,an inhibitor of PKA,attenuated the decrease in respiratory rate caused by fentanyl.Adrenergic receptors may be involved in fentanyl-induced respiratory depression,and NMDA receptor-assisted agonist D-serine is not involved in fentanyl-induced respiratory depression.3.Epac participates in fentanyl-induced respiratory depression and its related mechanismsEpac agonist 8-pCPT（10,20μg/site,i.c.v）can significantly reduce the respiratory rate caused by fentanyl(F_2,120=4.70,P<0.05),reducing the inspiratory time(F_2,120=6.39,P<0.01)and expiratory time(F_2,120=8.55,P<0.01),increasing the minute ventilation(F_2,120=4.16,P<0.05).MEK inhibitors U0126 and SL327 significantly increased the respiratory rate of animals treated with fentanyl(F_2,96=16.41,P<0.001),reducing the inspiratory time of animals(F_2,96=7.86,P<0.01)and expiratory time(F_2,96=21.70,P<0.001).SP600125 and U0126 did not affect morphine-induced respiratory depression in mice（P>0.05）.8-pCPT pretreatment reduced the ERK phosphorylation level induced by fentanyl in CHO-μcell line and increased the phosphorylation level of ERK induced by morphine.The intracellular distribution of phosphorylated ERK in PC12 cells was different between morphine and fentanyl.The phosphorylated ERK was mainly distributed in the nucleus after fentanyl treatment.The phosphorylated ERK was mainly distributed in the cytoplasm after morphine treatment.Conclusions:Unlike morphine,β-arrestin 2 is not involved in fentanyl-induced respiratory depression,which may be due to the different duration and intracellular distribution of p-ERK after morphine and fentanyl treatment.Both Epac and PKA are involved in the respiratory depression of fentanyl,and they have the opposite effect.The mechanisms by which Epac alleviates the fentanyl-induced respiratory depression may be related to inhibition of intracellular ERK phosphorylation.