The Effect Of I₁-imidazoline Receptor On The Function Of ?_2A-adrenergic Receptor And Its Mechanism

The imidazoline receptors?IRs?is a kind of novel receptor discovered by Bousquet et al.in studying mechanisms underlying the central antihypertensive effects of clonidine in the mid-1980s.It was divided into three subtypes,which are I₁R,I₂R and I₃R.I₁R is related to various physiological functions and pathological processes such as inhibiting drug addiction,hypotension,anti-depression,anti-anxiety and promoting cognition.I₁R is closely related to the alpha 2 adrenergic receptor??₂AR?.However,due to the absence of specific agonist or antagonist of I₁R-drugs acting on I₁R can also bind to?₂AR,and the similar distribution of I₁R and?₂AR in tissue and cells,the relationship between I₁R and?₂AR remained unclear,and limited the further functional study on I₁R and?₂AR.?₂AR is an important class of G-protein-coupled receptor,and can be divided into?_2AAR,?_2BAR and?_2CAR subtypes.Although?₂AR agonist dexmedetomidine?DEX?and antagonist yohimbine?YOH?have no subtype selectivity,it has been proven that?_2AAR mediated?₂AR-related analgesia and anesthesia by using?_2AAR knockout mice.Therefore,DEX and YOH are good tools to study the analgesic and anesthetic effects of?_2AAR.In this study,using previously established I₁R knockout mice and DEX as tools,we studied the effect of I₁R on the biological function of?_2AAR in animal models including 56?hot plate,tail-flick,formalin test and loss of righting reflex.In addition,we investigated the molecular mechanism underlying the regulation of I₁R on biological functions of?_2AAR in vitro using established CHO cells stably expressing mouse I₁R,?_2AAR,and co-expressing mouse I₁R and?_2AAR,respectively.Our findings are as follows:?1?In the 56 ^oC hot plate test,after 30 min treatment with DEX?40?g/kg,i.m.?,the latency of licking paws of wildtype mice was 46.15±4.66 s,and the percentage of maximum possible analgesic effect was 66.57±11.60%;while the latency of licking paws in I₁R knockout mice was 25.90±5.28 s,and the percentage of maximum possible effect was 19.88±14.31%.Compared with the wildtype mice,the latency of licking paws and percentage of maximum possible analgesic effect were significantly shortened?P<0.001,n=9-11?and reduced?P<0.01,n=9-11?.In the tail-flick test,after 30 min treatment with DEX?40?g/kg,i.m.?,the tail-flick latency was significantly shortened form 60 s?wildtype?to 11.64±0.77s?knockout??P<0.001,n=12?,and the percentage of maximum possible analgesic effect was significantly reduced from 100%?wildtype?to 0.77±1.86%?knockout??P<0.001,n=12?.In the formalin test,after 30 min treatment with DEX?40?g/kg,i.m.?,mice licking time was significantly extended from 21.86±11.17 s?wildtype?to 94.38±14.87 s?knockout??P<0.05,n=8-9?.The above results indicated that absent of I₁R significantly downregulated the analgesic effect of DEX.?2?In the mice hot plate test,pretreatment with high selective?₂AR blockade yohimbine?1.5 or 3.0 mg/kg,i.m.?totally eliminated the analgesic effect of DEX,suggesting that the analgesic effect of DEX may be mediated by activating of?_2AAR rather than I₁R?as described above?.?3?In the loss of righting reflex,after treatment with DEX?800?g/kg,i.m.?,the loss of righting induction time was significantly extended from 0.73±0.21 h?wildtype?to 2.63±0.40 h?knockout??P<0.001,n=8-9?;the loss of righting duration time was significantly shortened from 10.77±0.74 h?wildtype?to 7.42±0.95 h?knockout??P<0.01,n=8-9?.1 h after treatment with DEX,the loss of righting percentage was significantly reduced from 77.8%?wildtype?to 0%?knockout??P<0.01,n=8-9?.These results indicated that absent of I₁R significantly downregulated the effect of DEX on the loss of righting.?4?On the basis of the above behavioral research,in order to further study the possible molecular mechanism underlying the enhancing biological effects of?_2AAR agonist DEX induced by I₁R,we established CHO cells stably expressing mouse I₁R alone?CHO-I₁R?,stably expressing mouse?_2AAR alone(CHO-?_2AAR),and stably co-expressing mouse I₁R and?_2AAR(CHO-I₁R/?_2AAR),respectively.In the saturation binding experiments of membrane protein preparation from CHO cells that expressed?_2AAR stably,the K_d and B_max values of³H-RX821002 were 0.96±0.24 nmol/L and 0.29±0.03 pmol/mg protein,respectively?n=3?,which were consistent with previous research.The majority of?_2AAR located on the membrane.In CHO cells,there were two forms of splicers,170 kD and 60 kD,both of which were distributed in membrane and cytoplasm.?5?In the above established cell models,we found that the expression of I₁R significantly increased the expression levels of?_2AAR?P<0.01,n=3?,and further upregulated DEX-induced ERK phosphorylation through activating?_2AAR?P<0.01,n=3?.These results suggested that I₁R-induced upregulation of DEX-activated?_2AAR may be related to its effects on increasing?_2AAR expression and/or facilitating its signal transduction processes.Conclusions:1.I₁R significantly enhanced?_2AAR function?analgesic and loss of righting reflex?;2.I₁R-induced upregulation of DEX-activated?_2AAR may be related to its effects on increasing?_2AAR expression and/or facilitating its signal transduction processes.