The Effects And Molecular Mechanism Of AT1R Biased Pathway For Catecholamine Secretion In Mouse Adrenal Chromaffin Cells

Objective:Angiotensin receptor 1（AT1R）is a typical G protein coupled receptor（GPCR）.and AT1R bias ligands produce different effects by activating G protein dependent or non-G protein dependent beta-arrestin pathways.Recent studies have shown that angiotensin-β-arrestin bias pathway for the treatment of heart failure,has a better effect than losartan.Angiotensin stimulate the adrenal gland to release adrenaline and norepinephrine.but adrenaline and norepinephrine is harmed for some of the cardiovascular disease.In particular,AT1R-arrestin bias-TRV 120027 has entered the third phase of clinical,so this research focused on the effect of arrestin-mediated AT1R ligands induce acute catecholamine secretion.Method:In this study.we used the specific G protein or beta-arrestin biased GPCR agonist and knockout mouse model to study the effect of beta-arrestin or G protein subtype-mediated GPCR on the angiotensin II receptor catecholamine secretion in the signaling type 1（AT1R）activates,which can mimic clinical conditions such as hypertension or a response to severe bleeding.We examined the effect of preclinical drug TRV120027 on catecholamine secretion on its effect on adrenal medulla function.In the study,pharmacological intervention was performed with the electrophysiological amperometric method in wild type or beta-arrestin-1（-/-）,beta-arrestin-2-/-,Gq-/-,TRPC3-/-knockout mice TRV120027 release of catecholamine mechanism.Result:1.G protein-biased AT1R agonists stimulate amperometric spikes of MACC Gq bias ligands TRV 120055（30 nM）and TRV 120056（50 nM）stimulated mouse adrenal chromaffin cells,causing the release of catecholamines.Statistical analysis revealed that the peak current of the Gq bias agonists（TRV 120055,TRV 120056）caused the secretion half of AngⅡ（100 nM）.2.β-arrestin-biased AT1R agonists stimulate acute catecholamine secretionβ-arrestin bias ligands SⅡ（1μM）,TRV120026（500 nM）and TRV120027（100 nM）stimulated mouse adrenal chromaffin cells,causing catecholamine secretion.Statistical analysis revealed that the peak current of the beta-arrestin-induced agonists（SⅡ,TRV 120027 and TRV 120026）was about half that of AngⅡ（100 nM）and that CA release by TRV 120027 could be completely inhibited by AT1R antagonist candesartan（100nM）.3.Different AT1R agonists induced epinephrine secretion in the adrenal medulla,measured by an Elisa Kit at 1min.In this study,we isolated intact mouse adrenal medulla,and detected the secretion of catecholamines（including adrenaline or norepinephrine respectively）by different ligands to stimulate in the case of ciliam,by ELISA kit.after stimulating 1min.We observed the increase of Adrenaline and norepinephrine.In the experiment,we isolated intact mouse adrenal medulla,simulated in vivo physiological conditions,respectively.detection of epinephrine（Epinephrine,E）and norepinephrine（Norepinephrine,NE）from medullary secretion caused by AT1R different bias ligand with ELISA kit.ATIR complete agonist Ang Ⅱ,AT1R Gq bias ligand（TRV120055）and AT1R P-catenin bias ligand（SII and TRV120027）short-term stimulation of 1min can lead to increased levels of medulla E and NE secretion.4.Amperometric spikes of chromaffin cells stimulated by G protein-biased AT1R agonists were blocked by the PLC inhibitor U73122.In the experiment,Ang II（100 nM）stimulated MACC to release catecholamine secretion,by 10uM U73122 blockade,secretion peak decreased,catecholamine secretion decreased about half percent.TRV 120055 and TRV120056 stimulation after many CA release,and then given U73122,TRV120055 and TRV120056 stimulation,almost no detection of catecholamines secretion,Statistical analysis,the result is statistically significant for the difference ofTRV120055 before and after the U73122 blocked.5.PLC inhibitor U73122 had no effect on the secretion of chromaffin cells stimulated by β-arrestin biased AT1R agonistsRespectively,1uM SII,100nM TRV 120027 and 500nM TRV 120026 to stimulate the MACC can be seen catecholamine secretion,after washing with external fluid 2min.after 10uM U73122 blocked,and then the secretion peak stimulated by the ligands is no significant changes,catecholamine secretion before and after the difference was not statistically significant in statistical analysis.From the above results we can find that(3-arrestin bias ligand stimulating the secretion of MACC can not be blocked by U73122.6.TRV 120027-induced amperometric spikes of chromaffin cells were independent of Gg pathways100nM TRV 120027 stimulated chromaffin cells from Gq-/-gene knockout mice,the results showed that catecholamine release of Gq knockout and WT MACC was no statistically significant difference.7.TRV 120027-induced amperometric spikes of chromaffin cells were independent of Gi pathwayIn the experiment,we selected the pertussis toxin（PTX）-Gi inhibitor,and then MACCs incubated with 1μM PTX.After MACCs were stimulated by 100nM TRV 120027,the quantities of monoamine neurotransmitters showed that there was no statistical significance in the single factor analysis of variance.7.Beta-arrestinl mediates TRV 120027 to stimulate MACC to cause catecholamine secretionThe chromaffin cells of beta-arrestin-2-/-gene knockout,stimulated by 100nM TRV120027 are well-secreted.The chromaffin cells of beta-arrestinl-/-gene knockout female mice with 100nM TRV120027 stimulation,no quantitative detection of catecholamines was detected,the difference-TRV 120027 vs control was statistically significant.8.β-arrestin-biased signaling stimulates secretion through calcium influx.S Ⅱ and TRV 120027 did not record secretion in non-Ca²⁺ bath,statistical analysis,the difference was significant,between the secretion of non-Ca²⁺ bath and the standard calcium ion solution.9.β-arrestin-1-biased signaling stimulates secretion through TRPC3 activationIn the study 100nM TRV 120027 TRPC3+/-TRPC6-/-TRPC7-/-can detect the secretion peak,while TRPC3-/-TRPC6-/-TRPC7-/-chromaffin cells did not detect secretion peaks,statistically analyzed,（P<0.005）.and it was further confirmed that TRV 120027 did not record the peak of the quantification of catecholamines after TRV 120027 stimulation,except that TRPC3-/-gene mice were knocked out.Activation of TRPC3 channel caused catecholamine secretion.10.The beta-arrestin-1-TRPC3 pathway is a distinct subset of seven transmembrane receptors in the general pathway of different GPCR agonists-induced secretion.Finally,to examine whether the β-arrestin-1-TRPC3 pathway underlies catecholamine secretion after activation of other G protein coupled receptors,we treated primary chromaffin cells with acetyl-β-methacholine chloride（Mch）.cholecystokinin-8s（CCK-8s）and oxytocin（OT）.which are known to activate the muscarinic acetylcholinc receptor（mAchR）.the cholecystokinin receptor（CCKAR/CCKBR）.and the oxytocin receptor（OXTR）,respectively.Application of the PLC inhibitor U73122 blocked the amperometric spikes by 80%and 50%for primary chromaffin cells stimulated with OT and CCK-8s,respectively.Pre-incubation of chromaffin cells with Pyr3 had no significant effect on either OT-or CCK-8s-induced spikes after U73122 application,indicating that TRPC3 was not involved in catecholamine secretion after activation of either the cholecystokinin receptor or OXTR.However,whereas U73122 reduced Mch-induced amperometric spikes by 50%,the combination usage of Pyr3 and U73122 further reduced Mch-induced catecholamine secretion to approximately 15%,suggesting that TRPC3 activation also underlies mAchR-mediated secretion in chromaffin cells.Consistently,Mch induced catecholamine secretion was significantly reduced in β-arrestin-1（-/-）mice compared to wild type mice.Application of U73122 significantly reduced Mch-induced catecholamine secretion by approximately 40%in wild type mice whereas it decreased Mch-induced catecholamine secretion by almost 80%inβ-arrestin-1（-/-）mice.Therefore,β-arrestin-l-TRPC3 pathway is an active component in mediating Mch induced catecholamine secretion in primary chromaffin cells.Taken together,these results suggest that the(3-arrestin-l-TRPC3 pathway underlies the acute catecholamine.DiscussionTaken together,we revised the conventional thinking of how GPCRs regulate channel activities and in doing so shed light on the clinical treatment of heart failure.This work revealed that in addition to the classic G protein-TRP channel axis.β-arrestin-1 can also mediate acute physiologic responses via direct binding to and thus activating TRPC3 channels.The newly identified AT1R-b-arrestin-1-TRPC3 mode of secretion should be a paradigm-shift work in the field of GPCR signaling and arrestin functions,and will have a broad implication in understanding cellular responses to environmental stimuli under both physiologic and pathological conditions.Conclusion:1.β-arrestin bias ligands stimulating mouse adrenal chromaffin cells can cause catecholamine quantification release.2.β-arrestin bias ligands stimulate mouse adrenal pheochromocytoma-induced catecholamine release mediated by beta-arrestinl.3.β-arrestin bias ligands stimulation of mouse adrenal chromaffin-induced catecholamine release mainly rely on the extracellular calcium influx.4.Activation of TRPC3 channel caused by calcium influx caused catecholamine secretion.5.β-arrestin-1-TRPC 3 pathway underlies the acute catecholamine secretion downstream of a distinct subset of seven transmembrane receptors.Object:Almost all of the life activities are controlled by Ca²⁺ Ca²⁺as an intracellular second messenger,in the cell regulation,including the secretion of’ a variety of physiological processes.The core role of calcium in the regulation of secretion is established.In the process of vesicles,Ca²⁺ is the key factor to trigger the final fusion.According to our previous study.β-arrestin bias ligand to stimulate mouse adrenal chromaffin-induced catecholamine release mainly through the extracellular calcium influx to achieve.So we studied the role of calcium channels in the changes of calcium ions induced by β-arrestin bias ligands.Method:In this study.calcium imaging was used to clearly demonstrate the changes and mechanisms of arrestin-mediated intracellular Ca²⁺ in catecholamine,and confirmed by changes in calcium in primary chromaffin cells of mice from β-arrestin2-/-,β-arrestin2-/-,TRPC3-/-knockout mice.And 293 cells were used to co-express AT1R-cherry.beta-arrestinl-RFP and TRPC3-GFP to verify TRV120027 The localization of AT1R,β-arrestinl and TRPC3 on the cell membrane after stimulation.Result:1.[beta]-arrestin bias ligand stimulates MACC-induced[Ca²⁺]_i can not be blocked by U73122In this study,100 nM AngⅡ stimulated MACCs increased F340/380 ratio,intracellular[Ca²⁺]_i increased,and then AngⅡ stimulated pheochromycin cell ratio decreased by about 50%by U73122 blocked.The same results were obtained in beta-arrestin-2-/-mice.And the ratio of F340/380 increased by about 0.4 after stimulation with 100 nM TRV120027 in P-arrestin-2-/-knockout mice,suggesting that β-arrestin-2 could not mediate TRV120027-induced chromaffin cells[Ca²⁺]_i.And there was no significant difference between the two groups about[Ca²⁺]_i（P<0.05）,which was consistent with the effect of the first part of the results on the secretion of chromaffin cells.2.β-arrestinl mediates TRV 120027-induced changes in[Ca²⁺]_i in MACCsIn the present study,the ratio of F340/380 increased with the expression ofβ-arrestin-1-/-knockout with 100 nM AngⅡ.the change of intracellular[Ca²⁺],was blocked with U73122.There was significantly difference in the ratio of chromaffin cells between the two groups（p<0.005）.Secondly,the ratio of F340/380 did not change by 100nM TRV 120027on MACCs from β-arrestin-1-/-mice,that is,the[Ca²⁺]_i did not change in MACCs.As a result,β-arrestin-1 mediates the[Ca²⁺]_i changes in chromaffin cells induced by TRV 120027.3.Effects of extracellular Ca²⁺ on[Ca²⁺]_i in TRV120027 on MACCsS Ⅱ and I RV120027 stimulated chromaffin cells in calcium-free solution,and no significant changes in[Ca²⁺]_i were recorded,and had statistically significant differences（p<0.01）.KCI on MACCs stimulates the increase in[Ca²⁺]_i,when it is switched to the outer liquid without calcium ions,nor is it detected.Statistical analysis showed significant differences（p<0.005）.It was confirmed that extracellular Ca²⁺ influx,caused by TRV 120027 on chromaffin cells[Ca²⁺]_i increasing.4.TRP channel mediated TRV 120027-induced changes in[Ca²⁺]_i in MACCsIn the experiment,different calcium channel blockers,L-type calcium channel blocker nicardipine,R-type calcium channel blocker SNX482 and voltage-gated calcium channel blocker CdCl2 were selected to block the corresponding channel,TRV 120027 on MACC still recorded to the F340/F380 ratio increasing,the statistical analysis inhibition rate was not significant,did not block the calcium influx.Finally,non-specific TRP channel blocker ruthenium red,blocking the TRP channel,and then 100nM TRV120027 stimulation of MACCs,did not appear the change of F340/F380 ratio,the statistical analysis of inhibition rate,calcium influx was blocked 5.TRPC3 mediates TRV120027-induced changes in[Ca²⁺]_i in MACCs In the experiment,different TRP channel blockers,TRPV4 specific blocking agent HC067047 and TRPC4 specific blocker ML-204 blocked the corresponding channel,TRV 120027 on MACCs still recorded F340/F380 ratio increasing,statistics Analysis of inhibition rate was not significant,did not block calcium influx.After the selection of nonspecific TRPC3/6/7 blocker lanthanum chloride,100nM TRV 120027 on the chromaffin cells,and the ratio of F340/F3 80 was not increased.The inhibition rate was obvious and the calcium influx was blocked.After application of specific TRPC3 channel blocker Pyr3.TRV120027 on MACCs calcium influx was also blocked.The above results suggest that TRV120027-induced increasing in[Ca²⁺]_i in the chromaffin cells is mediated by the TRPC3 channel.6.TRPC3.beta-arrestin-1 and ATI R co-cxpressionThe TRPC3-GFP and β-arrestin-1-RFP co-transfected 293 cells,100nM Ang Ⅱand 100nM TRV 120007 stimulated β-arrestin-1-RFP on the membrane,Merge and TRPC3 co-localized on the cell membrane.TRPC3-GFP and AT1R-cherry were co-transfected into 293 cells.100nM Ang Ⅱ and 100nM TRV 120027,and the posterior plate of ATI R receptor was localized.Merge was co-localized with TRPC3 on the cell membrane.Confirmed TRV120027 stimulation of ATI R-arrestin-1 after rapid conjugation activates TRPC3 channel.p-arrestin-mediated coupling of AT1R to TRPC3 is a novel way of GPCR activation.Discussion:The results indicate that AT1R recruits b-arrestin-1 to the plasma membrane and promotes the formation of AT1R-b-arrestin-1-TRPC3 complex in a quick manner,thus leading to the direct opening of TRPC3 channels.which then mediates extracellular Ca2C influx and triggers catecholamine secretion.Conclusion:1.β-arrestin-1 mediates TRV 120027-induced changes in[Ca²⁺]_i in MACCs2.TRPC3 mediates TRV120027-induced changes in[Ca²⁺]_i in MACCs.