The Role And Mechanism Of Rostral Ventrolateral Salusin-β In The Pathogenesis Of Hypertension

BackgroundSalusins are translated from an alternatively spliced mRNA of torison dystonia (TOR2A), a gene encoding a protein of the torsion dystonia family and composed of two related bioactive peptides of 28 and 20 amino acids designated salusin-a and salusin-β. Salusin-β is shown to have negative inotropic effect on ventricular muscle. Salusin-β-like immunopositive cells have been found in suprachiasmatic, supraoptic and paraventricular nucleus (PVN), and salusin-positive nerve fibers and their terminals in the posterior pituitary. A recent study in our lab has shown that salusin-P in the PVN increases renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) in two-kidney, one-clip (2K1C)-induced hypertensive rats via both circulating AVP and AVP in rostral ventrolateral medulla (RVLM). RVLM contributes to the elevated blood pressure in a number of hypertension models. Microinjection of AVP V1 receptor antagonist (dTyr(CH2)5(Me)AVP, AAVP) into the RVLM abolished the effects of salusin-β in the PVN on the RSNA and HR but only attenuated the effects of salusin-β on the MAP. However, it is unknown whether salusin-P is expressed in the RVLM, and whether salusin-β in the RVLM is invoved in cardiovascular regulation in hypertension rats. The present study was designed to determine the roles and the downstream molecular mechanism of salusin-β in the RVLM in sympathetic activation and hypertension in renovascular hypertensive rats.Objective1 To investigate the expression of salusin-β in the RVLM.2 To investigate whether salusin-β in the RVLM of hypertension rats have regulatory role on sympathetic activity and blood pressure.3 To investigate the downstream molecular mechanism of salusin-β in sympathetic activation and blood pressure.MethodsThe experiment used the two-kidney, one-clip (2K1C) method to make the renal vascular hypertension rat model, the control group rats subjected to the same operation, except for the U-shaped clip was not used. Tail artery systolic pressure greater than or equal to 160 mmHg is set as successful modeling standards. With immunohistochemical method to study the expression of salusin-β immunoreactive positive cells in the RVLM. Using immunofluorescence double staining to determine which cell types that salusin-P expressed in the RVLM. Acute experiments were carried out 4 weeks after the 2K1C or sham operation (Sham) under urethane and alphachloralose anesthesia. Using PowerLab signal acquisition system to recording the renal sympathetic nerve activity (RSNA), mean arterial blood pressure (MAP) and heart rate (HR) under the conditions of mechanical ventilation. Using rat brain stereotaxic instrument to position and bilateral microinjection of drugs into the RVLM. Salusin-p level in the RVLM was mesured by enzyme-linked immunosorbent assay (ELISA), as well as AVP levels in the plasma and RVLM. Using chemiluminescent assay to determine the superoxide anion level and NAD(P)H oxidase activity in RVLM. In-situ detection of superoxide anions produced in the RVLM was determined by dihydroethidium (DHE) fluorescent probe.Results1. Compaired with Sham rats, salusin-β-like positive cells distributed in RVLM and the number of salusin-β-like positive cells in the RVLM of 2K1C rats was significantly increased.2. Salusin-β level in the RVLM of 2K1C rats also higher than that of Sham rats when determined by ELISA.3. Cells co-location technology of immunofluorescence double staining detected that the fluorescence of salusin-β in the RVLM is overlapped with the fluorescence of neuronal nuclear antigen (NeuN).4. Microinjection of different doses of salusin-β into the RVLM of Sham rats had no significant effect on RSNA or MAP, while microinjection of different doses of salusin-β increased RSNA, MAP and HR in a dose-related manner in 2K1C rats.5. Microinjection of anti-salusin-β IgG (SIgG) into the RVLM of sham rats failed to cause any significant effects on the baseline RSNA or MAP, while microinjection of SIgG into the RVLM of 2K1C rats decreased the baseline RSNA and MAP. Pretreatment with microinjection of CIgG into the RVLM of 2K1C rats had no significant effects on the effects of microinjection of salusin-β on the RSNA, MAP or HR. While pretreatment with microinjection of SIgG into the RVLM almost abolished the effects of salusin-β in the RVLM on the RSNA, MAP and HR in 2K1C rats. Since no significant effect of salusin-β or SIgG was observed in Sham rats, following experiments were only carried out on 2K1C rats.6. Microinjection of superoxide anion scavengers PEG-SOD or tempol, or NAD(P)H oxidase inhibitor apocynin into the RVLM reduced the baseline RSNA, MAP and HR in 2K1C rats. The RVLM pretreatment with PEG-SOD, tempol or apocynin almost abolished the RSNA, MAP and HR responses to salusin-β in the RVLM in 2K1C rats.7. Microinjection of salusin-P into the RVLM increased the superoxide anion level and NAD(P)H oxidase activity in the RVLM in 2K1C rats, which can be prevented by the pretreatment with SIgG. Microinjection of SIgG into the RVLM had a tendency in reducing the superoxide anion level and NAD(P)H oxidase activity in the RVLM, but the effects did not reach a significant level.8. Compared with the RVLM microinjection of saline, salusin-P increased the fluorescence intensity in the RVLM which detected by fluorogenic probe DHE in 2K1C rats.9. Microinjection of AAVP into the RVLM had no significant effects on the baseline RSNA, MAP and HR, as well as the RSNA, MAP and HR responses to salusin-β in the RVLM in 2K1C rats.10. Intravenous infusion of AAVP decreased the baseline RSNA, MAP and HR in 2K1C rats, but had no significant effects on the RSNA, MAP and HR responses to salusin-β in the RVLM.11. Microinjection of salusin-β into the RVLM had no significant effect on plasma AVP level or AVP level in the RVLM in 2K1C rats compared with saline. Similarly, SIgG had no significant effect on the plasma AVP level or AVP level in the RVLM in 2K1C rats compared with CIgGConclusionCompaired with the sham rats, the number of salusin-β positive neurons and the level of salusin-β were increased in the RVLM of renovascular hypertensive rats. Microinjection of salusin-β into the RVLM of sham rats had no significant effect on the RSNA and MAP, while microinjection of salusin-P into the RVLM of 2K1C rats could increases sympathetic outflow, blood pressure and heart rate. Salusin-β in the RVLM increases sympathetic outflow, blood pressure and heart rate via NAD(P)H oxidase-derived superoxide anions rather than by AVP or its V1 receptor in the RVLM and peripheral tissues in renovascular hypertensive rats.