Salusin-β In Paraventricular Nucleus Increases Blood Pressure And Sympathetic Outflow Via Vasopressin In Hypertensive Rats

BackgroundSalusins are originally identified from full-length human cDNAs bybioinformatics analyses in2003. They are translated from an alternatively splicedmRNA of human torsion dystonia-related gene (TOR2A), a gene encoding a proteinof the torsion dystonia family. Two related peptides salusin-α and salusin-β inhuman are identified, comprising28and20amino acids, respectively. Intravenousadministration of salusin-α and salusin-β to rats causes rapid, profound hypotensionand bradycardia. Salusin-β-like immunoreactivity was detected strongly in thehypothalamus and posterior pituitary, and less abundantly in anterior pituitary andgastrointestinal, immune, and hematopoietic systems. Paraventricular nucleus (PVN)is an important integrative site in the control of cardiovascular activity andsympathetic outflow via its projections to the intermediolateral column of spinalcord and the rostral ventrolateral medulla (RVLM). The PVN plays a role inregulating several sympatho-excitatory reflexes such as cardiac sympathetic afferentreflex, and adipose afferent reflex. The PVN is known to be very important insympathetic activation and hypertension in2K1C hypertensive rats, spontaneouslyhypertensive rats (SHR), and obesity hypertensive rats. Immunohistochemicalanalysis of rat tissues showed the presence of salusin-β in the PVN, and most of thesalusin-like immunoreactivity was detected in vasopressin-but not inoxytocin-containing neurons. Salusin-β stimulates the arginine vasopressin (AVP)release from perifused rat pituitary. The present study was designed to determinewhether salusin-β in PVN contributes to the hypertension and sympathetic activation in renovascular hypertensive rats, and whether the AVP is involved in the effects ofsalusin-β.Objective1. To determine whether salusin-β in the PVN modulate sympathetic activity orblood pressure in2K1C rats.2. To determine whether AVP is involved in the roles of salusin-β inhypertension and sympathetic activation.MethodsRenovascular hypertension was induced by two-kidney, one-clip (2K1C) in maleSprague–Dawley rats. The sham-operated rats (Sham) received similar surgery exceptthe clip was not used. The criterion of hypertension is set as systolic arterial pressure(SBP) of tail artery was greater than or equal to160mmHg in a conscious state.Acute experiments were carried out four weeks after2K1C or sham-operation underurethane and alpha-chloralose anesthesia. The renal sympathetic nerve activity(RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded bypowerlab biological signal acquisition system. The enzyme-linked immunosorbentassay (ELISA) was used to meaure the plasma AVP, norepinephrine (NE) and Ang IIlevels. Immunohistochemical analysis was used to detect the presence of salusin-β inthe PVN.Protocol1: To determine the different doses of salusin-β on the RSNA, MAP andHR, either Sham or2K1C rats were randomly subjected to the PVN microinjection ofsaline,0.1,1,10or100pmol of salusin-β (n=6for each group). The intervalsbetween injections were at least40min for a complete recovery.Protocol2: To determine the effects of endogenous salusins, anti-salusin-β IgGwas used to immunoneutralize salusins in the PVN. Either Sham or2K1C rats wererandomly divided into four groups (n=6for each group), which were subjected to the PVN microinjection of control IgG (100ng), anti-salusin-β IgG (100ng), salusin-β(100pmol) pretreated with control IgG, or salusin-β pretreated with anti-salusin-β IgG.Salusin-β was administered5min after the pretreatment.Protocol3: Salusin-β-like immunoreactivity in the PVN was investigated inSham and2K1C rats (4sections for each rat, and3rats for each group).Protocol4: To determine the effects of salusin-β in the PVN on the sympatheticactivation, AVP release and Ang II production, either Sham or2K1C rats weresuccessively and randomly subjected to the PVN microinjection of saline or salusin-β(100pmol). The intervals between injections were at least40min for a completerecovery (n=6for each group). The blood samples were obtained10min after themicroinjection for norepinephrine, AVP and Ang II measurements.Protocol5: To determine whether peripheral AVP V1-receptors mediate theeffects of salusin-β in the PVN,2K1C rats were randomly divided into four groups(n=6for each group), which were subjected to the intravenous injection of saline (20μl/min) or dTyr(CH2)5(Me)AVP (AAVP, an AVP V1-receptor antagonist,4.0nmol/min), or intravenous infusion of saline or AAVP plus the PVN microinjection ofsalusin-β (100pmol). Salusin-β was administered5min after the intravenousinfusion.Protocol6: To determine whether AVP V1-receptors in the PVN mediate theeffects of salusin-β in the PVN,2K1C rats were randomly divided into four groups(n=6for each group), which were subjected to the PVN microinjection of saline orAAVP (40pmol), or the PVN microinjection of salusin-β (100pmol) pretreated withPVN microinjection of saline or AAVP. Salusin-β was administered5min after thepretreatment.Protocol7: To determine whether AVP V1-receptors in the RVLM mediate theeffects of salusin-β in the PVN,2K1C rats were randomly divided into four groups (n=6for each group), which were subjected to the RVLM microinjection of saline orAAVP (40pmol), or the PVN microinjection of salusin-β (100pmol) pretreated withRVLM microinjection of saline or AAVP. Salusin-β was administered5min after thepretreatment.Protocol8: To determine whether AT1receptors mediate the effects of salusin-βin the PVN,2K1C rats were randomly divided into four groups (n=6for each group),which were subjected to the intravenous injection of saline (20μl/min) or losartan(AT1receptor antagonist,150nmol/min), or intravenous infusion of saline or losartanplus the PVN microinjection of salusin-β (100pmol). Salusin-β was administered5min after the intravenous infusion.Results1. At the end of the4th week, SBP and MAP in2K1C rats were significantlyhigher than those in Sham rats, but no significant difference in body weight or HRwas found between Sham and2K1C rats.2. Microinjection of salusin-β into the PVN increased RSNA, MAP and HR in adose-related manner in2K1C rats, but not in Sham rats. There was a significant linearcorrelation between the dose of salusin-β and RSNA, MAP or HR change.3. Microinjection of anti-salusin-β IgG into the PVN decreased the baselineRSNA and MAP, and almost abolished the effects of salusin-β in the PVN on RSNA,MAP and HR in2K1C rats. No significant effects were found in Sham rats.4. Compared with Sham rats, the number of salusin-β-like immunopositiveneurons in the PVN was significantly increased in2K1C rats.5. The plasma norepinephrine, AVP and Ang II levels were higher in2K1C ratsthan Sham rats. Microinjection of salusin-β into the PVN further increased theplasma norepinephrine and AVP levels in2K1C rats, but not in Sham rats. However,salusin-β had no significant effect on plasma Ang II level in both Sham and2K1C rats.6. Intravenous injection of AVP V1-receptor antagonist AAVP decreased thebaseline RSNA and MAP, and abolished the effects of salusin-β in the PVN. However,intravenous injection of AAVP had no significant effects on baseline HR in2K1C rats.Intravenous injection of AT1receptor antagonist losartan decreased the baselineRSNA, MAP and HR, but failed to attenuate the effects of salusin-β in the PVN.7. Microinjection of AAVP into the PVN had no significant effects on thebaseline RSNA, MAP or HR as well as the effects of salusin-β in the PVN.8. Microinjection of AAVP into the RVLM had no significant effects on thebaseline RSNA, MAP or HR, but abolished the effects of salusin-β on RSNA and HRin the PVN.ConclusionsSalusin-β in the PVN increases blood pressure, heart rate and sympatheticoutflow via both circulating AVP and AVP in the RVLM in hypertensive rats.