| Angiotensin II and AT1 Receptors in the Paraventricular Nucleus Involved in the Enhanced Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive RatsBackgroudSympathetic over-excitation plays an important role in the pathogenesis of hypertension and the progression of organ damage. Inhibition of the enhanced sympathetic activity has been considered as an antihypertensive strategy. The cardiac sympathetic afferent reflex (CSAR) is a sympatho-excitatory reflex to increase sympathetic outflow and arterial pressure. The CSAR can be induced by stimulating the sympathetic afferent endings innervating the heart with epicardial application of capsaicin, bradykinin, adenosine. Our previous studies have shown that the CSAR was enhanced in two kidney-one clip (2K1C) rats. Central angiotensin II (Ang II) is an important modulator of the CSAR. The PVN is an important integrative site within the brain to control cardiovascular function. It is known that the PVN contains neurons that project to the intermediolateral cell column of the thoracolumbar spinal cord and the rostral ventrolateral medulla (RVLM) areas involved in controlling sympathetic nerve activity and blood pressure. We hypothesis that Ang II and angiotensin type 1 (AT1) receptor within the PVN involved in the enhanced cardiac sympathetic afferent reflex in renovascular hypertensive rats. To test the hypothesis, we investigated the effects of microinjection of Ang II and AT1 receptor antagonist losartan into the PVN on the CSAR and determined AT1 receptor expression in the PVN and RVLM in renovascular hypertensive rats and sham-operated rats.Objective 1. To determine Ang II in the PVN involved in the enhanced central gain of the CSAR in 2K1C rats.2. To investigate receptor mechanism of Ang II in the PVN involved in the enhanced central gain of the CSAR in 2K1C rats.MethodsExperiments with male Sprague-Dawley (SD) rats, weighing 250-300g and clean class, which came from Nanjing Medical University Laboratory Animal Center.The renovascular hypertension was induced with two-kidney one-clip (2K1C) method. Briefly, the rat was anesthetized with pentobarbital sodium (50 mg/kg, ip). A retroperitoneal flank incision was performed with sterile techniques. The right renal artery was exposed, and partially occluded by placing a silver clip with an internal diameter of 0.20 mm on the vessel to induce hypertension. The rats were kept in a temperature-controlled room on a 12-hour light and dark cycle with free access to standard chow and tap water. The sham-operated (Sham) rats were treated the same as the 2K1C rats except that their renal arteries were not occluded. The systolic arterial pressure (SBP) of tail artery was monitored with a PowerLab tail-cuff system (ADInstruments, Australia). The criterion of hypertension in the present study is set as SBP>160 mm Hg. At the end of the fourth weeks, the acute animal experiments were carried out in anesthetized rats. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) were recorded in vivo on a PowerLab data acquisition system have done after four weeks. The CSAR was evaluated by the RSNA response to epicardial application of capsaicin. The coordinates for PVN were determined in a stereotaxic instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The rats were euthanized with an overdose of pentobarbital. The brains were removed and quickly frozen with liquid nitrogen and stored at ?70°C until being sectioned. The serial sections in the level of coronal were cut through the PVN and RVLM levels, respectively. The PVN and RVLM areas were punched out with a 10-gauge needle (inner diameter 1.0 mm). The AT1 receptor protein of the PVN and RVLM were measured with the Western blotting.1. The effects of PVN microinjection of ACSF and different doses of Ang II (0.03, 0.3 and 3 nmol) on the CSAR, the baseline RSNA and MAP were determined in in Sham and 2K1C rats respectively (n=6 for each group).2. The effects of losartan on the CSAR, the baseline RSNA and MAP were determined. PVN microinjections of ACSF (n=6), Ang II (3 nmol), losartan (50 nmol) and losartan (50 nmol) plus Ang II (3 nmol) were carried out in Sham and 2K1C rats respectively. The pretreatment with losartan was carried out 10 minutes before Ang II. The CSAR was determined after the PVN microinjection (n=7 for each group).To exclude the possibility that the effects of Ang II on the CSAR were caused by diffusion to other brain area, the effects of microinjection of Ang II (3 nmol) into the anterior hypothalamic area which is the adjacent areas of the PVN were determined (n=3).3. The AT1 receptor protein expression of the PVN and RVLM in Sham and 2K1C rats were determined. Sham and 2K1C rats were euthanized with an overdose of pentobarbital. The brains were removed and quickly frozen with liquid nitrogen and stored at ?70°C until being sectioned. The serial sections in the level of coronal were cut through the PVN and RVLM levels, respectively. The PVN and RVLM areas were punched out with a 10-gauge needle (inner diameter 1.0 mm). The punched tissues were put in 0.1 mL of lysis buffer and homogenized. The total protein in the homogenate was extracted and measured using protein assay kit (BCA, Pierce).AT1 receptor protein in the PVN and RVLM were measured with the Western blotting.Results1. Microinjection of Ang II into the PVN dose-relatedly increased the CSAR, baseline RSNA and MAP in Sham and 2K1C rats. After administration of Ang II, the CSAR was greater in 2K1C rats than Sham rats. Microinjection of Ang II into the anterior hypothalamic area which is the adjacent areas of the PVN failed to cause any significant effect on the CSAR.2. PVN microinjection of losartan decreased the CSAR, RSNA and MAP in 2K1C rats, but had no significant effect on the CSAR, RSNA and MAP in Sham rats. Pretreatment with losartan abolished the enhanced CSAR, RSNA and MAP effect caused by Ang II in both Sham and 2K1C rats.3. Compared with Sham rats, the AT1 receptor expression in both PVN and RVLM increased significantly in 2K1C rats . ConclusionsMicroinjection of Ang II into the PVN enhanced the CSAR in both 2K1C and Sham rats. The Ang II-induced enhancement in CSAR was significantly greater in 2K1C rats than Sham rats. The effects of Ang II on CSAR were mediated by AT1 receptors. The AT1 receptor antagonist losartan normalized the enhanced CSAR in 2K1C rats.Part 2Endothelin-1 in Paraventricular Nucleus Modulates Cardiac Sympathetic Afferent Reflex in RatsBackgroundThe cardiac sympathetic afferent reflex (CSAR) is a positive feedback sympathetic excitatory reflex which plays a critical role in the pathogenesis of CHF and hypertension. The paraventricular nucleus (PVN) is an important integrative region in the control of sympathetic outflow, arterial pressure and CSAR. However the molecular mechanism of CSAR mediated by PVN is not very clear. It has been found that the reactive oxygen species (ROS) especially superoxide anions in the PVN mediate the CSAR, and that endothelin-1 (ET-1) activates NADPH oxidases and thereby increasing superoxide production and resulting in oxidative stress and cardiovascular dysfunction, and that the lesion of the PVN preventes the ET-1 induced increase in arterial pressure and sympathetic activity. We hyperthesis that ET-1 in the PVN involved in the regulation of CSAR, which mediated by the ETA receptor and the molecular mechanism of regulation may be related to superoxide anion. To test our hypothesis, we investigated the effects of microinjection of ET-1, ETA receptor antagonist BQ-123, super oxygen anion scavenger tempol and PEG-SOD into the PVN on the CSAR evoked by epicardial application of capsaicin. Furthermore, the superoxide anion levels in the PVN were determined.Objective1. To determine the effects of of ET-1 in the PVN on the CSAR, sympathetic outflow and MAP.2. To investigate the molecular mechanism of ET-1 in the PVN on the CSAR, sympathetic outflow and MAP.MethodsIn anesthetized rats with sinoaortic denervation and cervical vagotomy, renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded in vivo on a PowerLab data acquisition system. The CSAR was evaluated by the RSNA response to epicardial application of capsaicin. The coordinates for PVN were determined in a stereotaxic instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The rats were euthanized with an overdose of pentobarbital. The rat brains were removed quickly, flash-frozen in liquid nitrogen and stored at ? 70°C. Coronal sections of the brain were made using a cryostat microtome (CM1900, Leica LTD), and the PVN area was punched out with a 10-gauge needle. The punched PVN tissue was homogenized and then centrifuged. Protein concentrations in the supernatant were measured with the Bradford assay. Lucigenin-derived chemiluminescence is a valid probe for detecting superoxide anion. The lucigenin reacts with the superoxide anion in the sample to yield an unstable dioxetane intermediate. The lucigenin dioxetane breaks down into two molecules of N-methylacridone, one of which is in an electronically excited state, which becomes the ground state by emitting a photon. The superoxide anion level is determined by measuring the photon emission. The reaction was started by addition of dark-adapted lucigenin (5?M). Light emission was measured with a luminometer (20/20n, Turner, CA), and values were expressed as mean light unit (MLU) per minute per milligram of protein, which represents the superoxide anion level. Background chemiluminescence in the buffer containing lucigenin (5?M) was measured.1. The effects of PVN microinjection of ACSF and different doses of ET-1 (0.1, 1 and 10 pmol) on the CSAR, the baseline RSNA and MAP were respectively determined in four groups of rats (n=6 for each group).2. The effects of BQ123 on the CSAR, the baseline RSNA and MAP were determined. PVN microinjections of ACSF (n=6), ET-1 (10 pmol, n=6), BQ123 (40 nmol, n=7) and ET-1 (10 pmol, n=7) pretreated with BQ123 (40 nmol) were carried out in four groups of rats. The pretreatment with BQ123 was carried out 15 minutes before ET-1. The CSAR was determined 10 minutes after the PVN microinjection.To exclude the possibility that the effects of ET-1 on the CSAR were caused by diffusion to other brain area, the effects of microinjection of ET-1 (10 pmol) into the anterior hypothalamic area which is the adjacent areas of the PVN were determined (n=3).3. The effects of superoxide anion scavenger, tempol, and PEG-SOD, on the enhanced CSAR and RSNA responses as well as pressor response induced by ET-1 were investigated. PVN microinjections of ACSF (n=6), tempol (20 nmol, n=8) and PEG-SOD (2 units, n=8) alone, as well as ET-1 (10 pmol) pretreated with ACSF (n=6), tempol (20 nmol, n=8), and PEG-SOD (2 units, n=8) were carried out in 6 groups of rats. The pretreatment was carried out 15 minutes before ET-1. The CSAR was determined 10 minutes after the PVN microinjection.4. The superoxide anion level was measured with lucigenin-derived chemiluminescence in four groups of rats (n=7 for each). The measurement was carried out 10 minutes after the PVN microinjection of ACSF, ET-1 (10 pmol), BQ123 (40 nmol), ET-1 (10 pmol) pretreated with BQ123 (40 nmol).Results1. Microinjection of ET-1 into the PVN induced increases in CSAR, baseline RSNA and MAP. However, microinjection of ET-1 into the anterior hypothalamic area which is the adjacent areas of the PVN failed to cause any significant effect on the CSAR.2. PVN microinjection of ETA receptor antagonist had no significant effect on the CSAR, RSNA and MAP, but pretreatment with ETA receptor antagonist abolished the enhanced CSAR, RSNA and MAP effect caused by ET-1.3. Microinjection of superoxide anion scavenger, either tempol or PEG-SOD into the PVN inhibited the CSAR, and decreased the baseline RSNA and MAP. Pretreatment with tempol or PEG-SOD abolished the effect of ET-1 on the CSAR, the baseline RSNA and MAP.4. ET-1 significantly increased the superoxide anion level in the PVN, but ETA receptor antagonist BQ123 had no significant effects on the uperoxide anion level. Pretreatment with BQ123 abolished the increased superoxide anion level in the PVN caused by ET-1.Conclusions1. ET-1 in the PVN enhances the CSAR, baseline RSNA and MAP which is mediated by ETA receptors.2. The superoxide anions are involved in the regulation of CSAR, baseline RSNA and MAP and mediated the effects of ET-1 in PVN on the enhances the CSAR, baseline RSNA and MAP. |
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