Alamandine Injected Into The Paraventricular Nucleus Increases Blood Pressure And Sympathetic Activation In Spontaneously Hypertensive Rats

BackgroundThe renin-angiotensin system(RAS)is a complex system that plays an important role in regulating the cardiovascular system and electrolyte balance.Sympathetic activity has been shown to be enhanced in several disorders,including in patients with essential hypertension,obesity-related secondary hypertension or in chronic kidney disease,as well as in hypertensive models,such as spontaneously hypertensive rats(SHRs)and two-kidney one-clip hypertensive rats.The hypothalamic paraventricular nucleus(PVN)regulates the blood pressure and sympathetic activity via its projections to the rostral ventrolateral medulla(RVLM)and the intermediolateral column of the spinal cord.The sequence of alamandine is very similar to Ang(1–7),differing only by the substitution of an alanine residue for an aspartate residue in the amino terminus.Alamandine can be formed either through the decarboxylation of Ang-(1-7)or through the hydrolysis of Ang A by ACE2.Alamandine has been demonstrated to produce a pressor effect when microinjected into the RVLM,and a depressor effect when microinjected into the caudal ventrolateral medulla(CVML).Objectives1.To investigate whether alamandine acts in the PVN to modulate blood pressure and sympathetic activation in SHRs and WKY rats.2.To investigate whether the cyclic AMP(cAMP)-protein kinase A(PKA)pathway is involved in the alamandine-related sympathetic activation and pressor response.ProtocolsExperiments were carried out in 12-week-old male normotensive Wistar-Kyoto(WKY)rats and SHRs.All procedures were approved by the Experimental Animal Care and Use Committee of Nanjing Medical University,and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals(NIH publication No.85-23,revised 1996).The rats were kept in a temperature-controlled room on a 12 h light–dark cycle with free access to standard chow and tap water.In anesthetized rats,Rats were mechanically ventilated with room air using a rodent ventilator.Renal sympathetic nerve activity(RSNA)and mean arterial pressure(MAP)were recorded by a Power Lab data acquisition system.The bilateral PVN microinjections were performed with stereotaxie instrument.1.WKY rats and SHRs were randomized into 5 groups(n = 6 for each).The effect of the PVN microinjections of saline,three doses of alamandine(4,40 and 400 pmol),Ang II(40pmol)on RSNA and MAP were documented.2.WKY rats and SHRs were randomly divided into 4 groups(n = 6 for each).The effect of the PVN microinjections of Mrg D inhibitor D-Pro7-Ang-(1-7)(50 pmol),c AMP(1 nmol),AC inhibitor SQ22536(2 nmol),PKA inhibitor Rpc AMP(1 nmol)on RSNA and MAP were documented.,3.WKY rats and SHRs were randomly divided into 4 groups(n = 6 for each).The effects of PVN microinjection of alamandine(40 pmol)after PVN microinjection of D-Pro7-Ang-(1-7)(50 pmol)5minutes and AMP(1 nmol),AC inhibitor SQ22536(2 nmol),PKA inhibitor Rp-c AMP(1 nmol)8 minutes on the RSNA and MAP were documented.Results1.Microinjection of three doses of alamandine(4,40 and 400 pmol)into the PVN dose-dependently increased the MAP and RSNA,both middle and high doses of alamandine significantly increased the MAP and RSNA in both WKY rats and SHRs.The effects of alamandine in SHRs were greater than those in WKY rats.Ang II in the PVN increased the MAP and RSNA in both WKY rats and SHRs;however,these effects were more pronounced in the SHRs than in the WKY rats.There were no significant differences between the responses of MAP and RSNA to alamandine and to Ang II.Alamandine in the PVN had no significant effect on heart rate in WKY rats and SHRs.2.Microinjections of the Mrg D inhibitor D-Pro7-Ang-(1-7)(50 pmol)decreased the RSNA and MAP in SHRs,but had no significant effect in the WKY rats.Pretreatment with D-Pro7-Ang-(1-7)in the PVN abolished the alamandinemediated effects on MAP and RSNA in both WKY rats and SHRs3.Microinjections of c AMP in the PVN observed an increase in the MAP and RSNA in both WKY rats and SHRs,however,c AMP induced greater increases of RSNA and MAP in the SHRs than in the WKY rats.Pretreatment of the PVN with c AMP enhanced the effects of alamandine on MAP and RSNA in both WKY rats and SHRs.4.Microinjections of the AC inhibitor SQ22536 decreased the RSNA and MAP in SHRs,but had no significant effect in the WKY rats.Pretreatment with SQ22536 in the PVN abolished the alamandine-mediated effects on MAP and RSNA in both WKY rats and SHRs5.Microinjections of the PKA inhibitor Rp-c AMP in the PVN decreased MAP and RSNA in SHRs,whereas it had no significant effect on the WKY rats.Pretreatment with Rp-c AMP in the PVN inhibited the effects of alamandine on the MAP and RSNA in both WKY rats and SHRsConclusion1.Administration of alamandine in the PVN increases blood pressure and sympathetic output by activating Mrg D.Alamandine exerts more pronounced effects in hypertensive rats than WKY rats.2.The c AMP-PKA pathway mediates alamandine’s effects in the PVN,suggesting a role for this pathway in high blood pressure and sympathetic activation in spontaneous hypertension.