?-Arrestinl1 In Sympathetic Center Participates In Cardiovascular Regulation In Hypertension

Part one:?-arrestinl in the RVLM downregulats AT1R in hypertension via downregulatingBackgrounds and ObjectivesEssential hypertension is a chronic genetic disease and a common cardiovascular dysfunction disease of human beings as well.It is occupying around 90 percent of the whole hypertension suffers.Until now,it is still vague about the pathogenesis of hypertension because of its complexity.Although hypertension could be well controlled by using antihypertensive drugs,the occurrence of severe complications like heart failure,stroke and kidney injury has always been at a high rank.We truly need put further endeavors to research into the pathogenesis of hypertension more comprehensively to find more effective precautionary and therapeutic strategies.Hypertension is characterized by sympathetic overactivity,which is closely involved in the prognosis of it.Rostral ventrolateral medulla(RVLM)serves as a central part switching the sympathetic activities and maintaining the physiological tone of blood pressure.It has been reported that excessive Ang? stimulates AT1R dependent G protein signaling pathway,evoking overloaded inflammation and oxidative stress in the RVLM,and leads to excitation of pre-sympathetic neurons in the RVLM and hypertension.Thus,blocking Ang II-ATR activity is an effective way to prevent hypertension.?-arrestinl is primarily regarded as a multi-functional cellular adaptor protein through its ability to mediate G protein coupled receptor(GPCR)desensitization and internalization.However,recent studies have revealed its extraordinary ability to inhibit G protein signals by ?-arrestin biased signaling pathway,and it seems to be cardiovascular protective.For example,carvedilol improves cardiomyocytes survival after heart failure by recruiting ?-arrestinl,and ?-arrestin1 is proved to protect neurons from apoptosis during stroke by activating Beclin-1 mediated autophagy reaction.?-arrestin1 may play a protective role in both peripheral and central cardiovascular system.However,it is still unclear whether ?-arrestinl is involved in the central regulation of sympathetic activity and blood pressure.Therefore,the first objective of this study is to determine the role of P-arresting in the RVLM in regulation of sympathetic activity and hypertension.In addition to its scaffolding function in the cytoplasm,?-arrestinl could also transfer into nucleus communicating with transcription factors such as NF-?B,which is one of the main promoters of AT1R gene expression.However,it still remains unmarked whether ?-arrestin1 is able to regulate NF-?B and AT1R expression,which is the second objective of this study.Furthermore,the third objective is to determine the role of ?-arrestin1 in preventing Ang II-AT1R induced hypertension.Hence,based on the backgrounds above,the main purpose of this study is to make it clear of the role of ?-arrestin1 in the RVLM in regulating sympathetic activity and hypertension.The results will provide solid support for clinic treatment of hypertension.Methods16w-years old male rats(WKY and SHR)were selected as the experiment animals.Animals were transfected by AAV in the RVLM and divided in four groups:WKY+AAR-GFP,WKY+AAV-Arrb1,SHR+AAV-GFP,SHR+AAV-Arrb1.The experimental rats were anaesthetized and the mean arterial pressure(MAP)and renal sympathetic nerve activity(RSNA)were monitored.After that,the expression of NF-?B and AT1R in the RVLM were detected by Western Blot.The AT1R function was then tested by microinjection of Ang II into the RVLM after ?-arresting overexpression.Results1.Overexpression of ?-arrestinl in the RVLM of SHR attenuated sympathetic hyperactivity and hypertension.The expression of ?-arrestin1 in the RVLM was significantly decreased in SHR compared with WKY,and it represented a decline with the growth of SHR.Next we conducted ?-arrestin1 overexpression in the RVLM,and the baseline MAP and RSNA were notably decreased compared with SHR with just GFP overexpression[Baseline MAP(mmHg):168±3.95 vs 138±3.06;Baseline Inte-RSNA(%Max):31.8±1.52 vs 22.6±2.73,P<0.05]?2.Overexpression of ?-arrestinl in the RVLM of SHR downregulated NF-?B phosphorylation and AT1R expression:SHR received p-arrestinl overexpression represented a significant decrease(around 66%)in AT1R expression in the RVLM compared with the control group.Meanwhile,the phosphorylation levels of NF-?B and I?Ba were similarly decreased by ?-arrestinl overexpression with a drop of 66%and 76%.In order to further determine the mechanism by which ?-arrestinl regulated NF-?B,immunoprecipitation helped us to certify the more tight interaction between ?-arrestinl and I?Ba of SHR post ?-arrestinl overexpression in the RVLM.This part of results suggested that ?-arrestinl overexpression in the RVLM downregulated AT1R expression by inhibiting NF-?B activation.3.Overexpression of ?-arrestinl in the RVLM prevented Ang ?-AT1R activationIn order to test the improvement of AT1R function,we conducted Ang?microinjection into the RVLM post ?-arrestinl overexpression,and the results showed that Ang ?-induced increases in MAP and RSNA of SHR were notably precluded by?-arrestinl instead of GFP[Change in MAP(%):9.61±.49 vs-5.85±3.5;Change in Inte-RSNA(%):31.59±4.8 vs-11.61±8.1,P<0.05];What's more,the similar inhibition was obtained in WKY[Change in MAP(%):19.3±1.5 vs 6.40±0.6;Change in Inte-RSNA(%):59.4±15.3 vs 20.2±5.1,P<0.05?.This part of results demonstrated an inhibition effect of P-arrestinl on Ang??AT1R axis.ConclusionOverexpression of ?-arrestinl in the RVLM attenuates hypertension by inhibiting NF-?B activation and AT1R expression,thus precluding excessive activation of Ang II-AT1R axis.The results will provide a new direction for well treating hypertension at the clinic.Part two:p-arrestinl raises NO release in the RVLM and the potential mechanismBackgrounds and ObjectivesNitric oxide(NO)is an important neuromodulator and cardiovascular modulator,and serves an important role on maintain physiological homeostasis of neuronal function and vasodilatation tone.NO is originated from L-Arginine catalyzed by NO synthases(NOS),which is consisted of three subtypes:nNOS,eNOS and iNOS.Foregoing narration has depicted complex and variable roles of different kinds of NOS and NO in the RVLM and NTS.For example,upregulation of nNOS/NO signaling in the RVLM significantly inhibits sympathoexcitation in rats with heart failure.Thus we can see the important role of NO on the regulation of neuronal activation both under physiological and pathological state.Evidence has proved that ?-arrestinl is critical for AKT mediated eNOS phosphorylation and NO release in human saphenous vein endothelial cells.What's more,?-arrestinl protects gastric mucosal cells against apoptosis via repressing iNOS in portal hypertensive gastropathy.It remains unclear the relationship between?-arrestinl and nNOS in the RVLM,although we have demonstrated the inhibitory effects of ?-arrestinl on hypertension and Ang II-AT1R axis in the part one.Thus,the main purpose of this part of study is to determine the role of ?-arrestinl in precessing of nNOS derived NO release in the RVLM.ERK1/2 is one of the most canonical biased signals recruited by ?-arrestinl,and is always active in cellular signaling transduction and physiological regulation of cell function.It has been demonstrated that overexpression of ?-arrestinl resulted in a significant increase in the cytosolic pool of phosphorylated ERK1/2 and a corresponding decrease in the nuclear pro-transcription activity following Ang II stimulation.ERK1/2 in the RVLM also plays an important role in the central control of blood pressure,but such a role seems to be effected by the upstream stimulators.For example,rapid stimulation of Ang II induced robust ERK1/2 phosphorylation in the RVLM followed by the acute pressor response.However,ERK1/2 underlies a hypotensive effect elicited by?za adrenergic receptor activation.In additional,ERK1/2 is responsible for neuron protection in brain stem death via upregulating nNOS activity.As for ?-arrestinl biased ERK1/2 signaling pathway in the RVLM on nNOS regulation,it is still unveiled and truly needs our further deep step to research into.MethodsAll the experimental animals were the same as that in part one.Total tissue NO content and NOS activity were detected by particular ELISA kits to test changes elicited by ?-arrestinl overexpression in the RVLM.Western Blot and immunofluorescence were used to detect the phosphorylation of nNOS and ERK1/2 in the RVLM.Then we conducted in-vivo siRNA transfection to knockdown ERK1/2 with a randomized interfering sequence as the contro1(CON siRNA),and the animals were then divided into four groups:AAV-GFP+CON siRNA,AAV-GFP+ERK1/2 siRNA,AAV-Arrbl+CON siRNA,AAV-Arrb1+ERK1/2 siRNA.Changes in baseline MAP and RSNA were monitored,nNOS phosphorylation and NO release were tested among the four groups.Results1.Overexpression of ?-arrestinl in the RVLM significantly upregulated nNOS-derived NO productionTotal RVLM tissue NO content and NOS activity were detected by particular ELISH kits.As a result,compared with AAV-Arrb1+CON siRNA group,NO content and NOS activity were significantly increased in AAV-Arrbl+ERK1/2 siRNA group with a rise of around 1.0 and 1.6 fold,respectively.Similarly,nNOS but eNOS phosphorylation levels were increased around 2.8 fold.2.Biased ERK1/2 signaling pathway mediated the ?-arrestinl-induced improvement in nNOS activity and NO releaseERK1/2 was knocked down in the RVLM by ERK1/2 siRNA post ?-arrestinl overexpression.The results showed that inhibition of ERK1/2 notably damaged the?-arrestin1-induced amelioration in MAP and RSNA compared with that without ERK1/2 inhibition[Baseline MAP(mmHg):145±2.6 vs 169±4.1;Baseline Inte-RSNA(%Max):19.7±1.3 vs 31.1±3.2,P<0.05].Meanwhile,nNOS activation and NO release by ?-arrestinl were also blocked by ERK1/2 suppression around 76%and 41%.ConclusionOverexpression of P-arrestinl in the RVLM promoted nNOS-derived NO release to ameliorating hypertension,and this was mediated by upregulating ?-arrestinl's ERK1/2 signaling pathway.This part of study had well established a bridge between ?-arrestinl and nNOS in the centra1 control of blood pressure,and supplied a stiff theoretical support for precluding and treating neuronal hypertension.