Role Of Reactive Oxygen Species In The Paraventricular Nucleus In The Pathogenesis Of Chronic Heart Failure And Renovascular Hypertension

It is well known that sympathetic activity is enhanced in chronic heart failure (CHF). This enhanced sympathetic activity contributes to further haemodynamic deterioration and the degree of sympathoexcitation is prognostic for survival in this disease. In CHF, cardiac sympathetic afferent reflex (CSAR) is over-activated and contributes to the increases in sympathetic outflow. Our previous studies have shown that angiotensin?(Ang?) and AT₁ receptors in the paraventricular nucleus (PVN) play an important role in the central modulation of CSAR, and contribute to the pathogenesis of enhanced CSAR in CHF. It has been reported that reactive oxygen species (ROS) are novel molecules involved in the intracellular signalling mechanisms of Ang?in the brain. We found that NAD(P)H oxidase-derived ROS in the PVN mediated the CSAR and contributed to the effect of Ang?in the PVN on the CSAR in normal rats.But whether the ROS in the PVN mediated the enhanced CSAR in CHF rats is still unknown. The present study was designed to investigate whether the ROS in the PVN is involved in the pathogenesis of enhanced CSAR in CHF rats, and whether ROS in the PVN mediates the Ang?-induced CSAR enhancement in CHF rats.1. To determine whether the ROS in the PVN is involved in the pathogenesis of enhanced CSAR in CHF rats.2. To determine whether the ROS in the PVN mediates the Ang?-induced CSAR enhancement in CHF rats.Experiments were carried out on male Sprague-Dawley rats weighing between 300 and 400 g. The rats were divided into two groups at random: CHF rats and sham rats. The CHF was induced by coronary artery ligation. The sham rats underwent the same operative procedure, but their coronary arteries were not ligated. The final experiment was carried out 6–8 weeks after coronary ligation or sham surgery. In 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 non infraction region application of bradinin (BK). The coordinates for PVN were determined in a stereotaxie instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The superoxide anions level in the PVN was measured with lucigenin-derived chemiluminescence method. Malondialdehyde (MDA) was used as an indirect marker of ROS level and was determined by the thiobarbituric acid spectrometric method.1.The effects of the PVN microinjection of saline, the superoxide anion scavengers, tempol (0.2, 2 and 20 nmol) or tiron (10 nmol) and superoxide dismutase inhibitor DETC (10 nmol) on the CSAR, baseline RSNA and MAP were determined in 6 groups of CHF rats and 6 groups of sham rats (n=6 for each).2. The effects of pretreatment with the PVN microinjection of saline, tempol (0.2, 2 and 20 nmol), tiron (10 nmol) and DETC (10 nmol) on the CSAR, RSNA and MAP responses to PVN microinjection of Ang?(0.3 nmol) were investigated in 6 groups of CHF rats and 6 groups of sham rats (n=6 for each).3. The effects of epicardial application of saline and BK (0.4?g) on MDA and superoxide anions levels in the PVN were determined in 2 groups of CHF rats and 2 groups of sham rats (n=6 for each).4. The effects of microinjection of saline, Ang?(0.3 nmol), losartan (10 nmol) and tempol (20 nmol) on MDA and superoxide anions levels in the PVN were determined in 4 groups of CHF rats and 4 groups of sham rats (n=6 for each).5. The effects of microinjection of tempol (20 nmol) into the anterior hypothalamic area, which is adjacent to the PVN, on the CSAR and Ang?-induced responses were determined in CHF and sham rats (n=3 for each).1. The mean infarct area was about 34% of the left ventricle in CHF rats, the heart weight and the heart-to-body weight ratio were increased in CHF rats. The left ventricle peak systolic pressure (LVSP), left ventricle developed pressure (LVDP) and left ventricle +dP/dtmax decreased, while left ventricle end-diastolic pressure (LVEDP) increased in CHF rats.2. Microinjection of the superoxide anion scavengers tempol or tiron into the PVN decreased baseline RSNA and MAP in both CHF and sham rats, but the DETC showed opposite effects. In addition, the RSNA change caused by DETC in CHF rats was greater than that in sham rats.3. The CSAR was enhanced in CHF rats. Microinjection of tempol or tiron into the PVN inhibited the CSAR, the highest dose of tempol abolished the enhanced CSAR. But DETC enhanced the CSAR.4. Microinjection of Ang?into the PVN increased RSNA and MAP in both CHF rats and sham rats, furthermore the RSNA response to Ang?in CHF rats was lager. Pretreatment with a high dose of tempol or tiron abolished the effects of Ang?on the RSNA and MAP in both CHF rats and sham rats. However, DETC did not further potentiate the effects of Ang?on RSNA and MAP.5. Microinjection of Ang?into the PVN significantly potentiated the CSAR in CHF rats. Pretreatment with microinjection of the highest dose of tempol or tiron into the PVN almost abolished the enhanced CSAR response induced by Ang?. While DETC augmented the enhanced CSAR response induced by Ang?in CHF rats.6. Microinjection of the highest dose of tempol into the anterior hypothalamic area (adjacent to the PVN) failed to inhibit the CSAR and the enhanced CSAR response induced by Ang?.7. The superoxide anions and MDA levels in the PVN were higher in CHF rats than that in sham rats. Microinjection of the highest dose of tempol decreased the superoxide anions and MDA levels in the PVN in CHF rats significantly.8. Epicardial application of BK increased MDA and superoxide anions levels in the PVN in both sham and CHF rats, furthermore the increased degree in CHF rats was larger.9. Microinjection of Ang?into the PVN increased superoxide anions and MDA levels in the PVN in both CHF and sham rats, but the increased degree in CHF rats was larger. The AT₁receptor antagonist losartan normalized the elevated superoxide anions and MDA levels in CHF rats.1. ROS in the PVN mediate the CSAR and the effect of Ang?in the PVN. 2. Increased ROS in the PVN is involved in the enhanced CSAR in CHF.It has been reported that sympathetic outflow is enhanced in patients with hypertension and in a variety of experimental hypertensive models. The elevated sympathetic activity contributes to the pathogenesis of hypertension and the progression of organ damage. Our recent study has shown that cardiac sympathetic afferent reflex (CSAR) is enhanced in 2K1C renovascular hypertensive rats and the enhanced CSAR in 2K1C rats partially contributes to the increased sympathetic outflow and hypertension. The central Ang?and AT₁receptors in the PVN play an important role in the enhanced central gain of the CSAR in 2K1C rats which can be attenuated by the PVN microinjection of ATB1B receptor antagonist losartan. It has been reported that reactive oxygen species (ROS) including superoxide anionsand hydroxyl radical, are novel molecules involved in the intracellular signalling mechanisms of Ang?in the brain. We found that NAD(P)H oxidase-derived superoxide anions in the PVN modulated the CSAR and contributed to the effect of Ang?in the PVN on the CSAR in normal rats and in chronic heart failure rats. But whether the ROS in the PVN mediated the enhanced CSAR in hypertensive rats is still unknown. This study was designed to determine whether ROS in the paraventricular nucleus (PVN) play role in modulating the CSAR and in mediating the enhanced effects of Ang?in the PVN in 2K1C renovascular hypertensive rats and investigate whether the NAD(P)H oxidase in the PVN is a key source of the ROS which modulates the CSAR and contributes to the effects of Ang?.1. To determine whether superoxide anions in the PVN play role on the enhanced CSAR in 2K1C hypertensive rats.2. To determine whether superoxide anions in the PVN play role on the effect of Ang?in the PVN in 2K1C hypertensive rats.3. To determine whether the NAD(P)H oxidase in the PVN was a key source of the ROS which modulated the CSAR and contributed to the effect of Ang?.Experiments were carried out on male Sprague-Dawley rats weighing between 300 and 350 g which were approved by the Experimental Animal Center of Nanjing Medical University. The rats were divided into two groups at random: renovascular hypertensive rats and sham rats. The renovascular hypertension was induced with two-kidney one-clip (2K1C) method. The sham rats underwent the same operative procedure excepet their renal arteries were not occluded. The final experiment was carried out 4 weeks after the 2K1C or sham surgery. In 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 stereotaxie instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The superoxide anions levels in the PVN were measured with lucigenin-derived chemiluminescence method. The NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescent detection of superoxide anions using a TD-20/20n Luminometer.1. The effects of PVN microinjection of saline, the superoxide anion scavengers tempol (20 nmol) and superoxide dismutase inhibitor DETC (10 nmol) on the CSAR, baseline RSNA and MAP were determined in 3 groups of 2K1C rats and 3 groups of sham rats (n=6 for each).2. The effects of PVN microinjection of 1%DMSO, NAD(P)H oxidase inhibitor apocynin(1 nmol) on the CSAR, baseline RSNA and MAP were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each)3. The effects of pretreatment with PVN microinjection of saline, tempol (20 nmol) and DETC (10 nmol) on the CSAR, RSNA and MAP responses to PVN microinjection of Ang?(0.3 nmol) were investigated in 3 groups of 2K1C rats and 3 groups of sham rats (n=6 for each).4. The effect of pretreatment with PVN microinjection of 1%DMSO, apocynin (1 nmol) on the CSAR, RSNA and MAP responses to PVN microinjection of Ang?(0.3 nmol) were investigated in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).5. The effects of epicardial application of saline and capsaicin (1 nmol) on superoxide anions levels and NAD(P)H oxidase activity in the PVN were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).6. The effects of microinjection of saline and Ang?(0.3 nmol) on superoxide anions levels and NAD(P)H oxidase activity in the PVN were determined in 2 groups of 2K1C rats and 2 groups of sham-perated rats (n=6 for each). 1. Microinjection of superoxide anions scavenger tempol or NAD(P)H oxidase inhibitor apocynin into the PVN significantly decreased, while SOD inhibitor DETC increased the basal RSNA, MAP in both 2K1C rats and sham rats. DETC caused greater response in basal RSNA in 2K1C rats compared with sham rats.2. The CSAR in 2K1C rats was enhanced. Microinjection of tempol or apocynin into the PVN significantly inhibited the enhanced CSAR, while DETC potentiated the enhanced CSAR in 2K1C rats.3. Microinjection of the Ang?into the PVN significantly increased the RSNA and MAP in both 2K1C rats and sham rats; furthermore the increased degree of RSNA in 2K1C rats was larger than that in sham rats. Pretreatment with PVN microinjection of tempol or apocynin attenuated the effects of Ang?in both 2K1C rats and sham rats. Pretreatment with DETC augmented the RSNA and MAP response induced by Ang?in 2K1C rats.4. Microinjection of the Ang?into the PVN significantly enhanced the CSAR in both 2K1C rats and sham rats; furthermore the increased degree of CSAR in 2K1C rats was larger than that in sham rats. Pretreatment with PVN microinjection of tempol or apocynin attenuated the effects of Ang?on CSAR in both 2K1C rats and sham rats. Pretreatment with DETC augmented the enhanced CSAR response induced by Ang?in 2K1C rats, but had no significant effect on the CSAR response in sham rats.5. The superoxide anions level and the NAD(P)H oxidase activity in the PVN were increased in 2K1C rats compared with sham rats.6. Microinjection of Ang?(0.3 nmol) into the PVN increased the superoxide anions level and NAD(P)H oxidase activity in the PVN in both 2K1C rats and sham rats compared with saline. Compared with sham rats, the effects of Ang?in 2K1C rats were greater.7. Epicardial application of capsaicin (1nmol) increased superoxide anions level and NAD(P)H oxidase activity in the PVN in both sham rats and 2K1C rats, furthermore these effects of epicardial application of capsaicin in 2K1C rats were greater than those in sham rats.1. ROS in the PVN mediate the CSAR and the effects of Ang?in the PVN.2. Increased superoxide anions level in the PVN is involved in the enhanced CSAR in hypertensive rats.3. NAD(P)H oxidase in the PVN was the major source of the ROS in modulating the CSAR and the effects of Ang?in the PVN.Our previous study has shown that cardiac sympathetic afferent reflex (CSAR) is enhanced in 2K1C rats and the enhanced CSAR contributes to the increased sympathetic outflow and hypertension. Central Ang?and AT₁receptors in the PVN play an important role in the enhanced CSAR in 2K1C rats. Recently we found that NAD(P)H oxidase-derived superoxide anions in the PVN mediated the enhanced CSAR and contributed to the effects of Ang?in the PVN in 2K1C rats. It has been reported that c-Src modulates the effect of Ang?in vascular smooth muscle cells through activating NAD(P)H oxidase and inducing ROS generation. C-Src is heavily expressed in the brain and in human neural tissues. Therefore we hypothesis that the c-Src in the PVN modulates the enhanced CSAR in 2K1C renovascular hypertensive rats through activating NAD(P)H oxidase and inducing ROS generation. This study was designed to determine whether c-Src in the paraventricular nucleus (PVN) play roles in regulating the CSAR and the enhanced effect of Ang?in the PVN in 2K1C renovascular hypertensive rats, and investigate the relationship between c-Src and reactive oxygen species (ROS) in the PVN in modulation of CSAR and the effects of Ang?in 2K1C renovascular hypertensive rats.1. To determine whether c-Src in the PVN plays a role in the enhanced CSAR in 2K1C hypertensive rats.2. To determine whether c-Src in the PVN is involved in the enhanced effects of Ang II in the PVN in 2K1C hypertensive rats.3. To investigat the relationship between c-Src and ROS in modulation of CSAR and the effects of Ang?in the PVN in 2K1C rats.Experiments were carried out on male Sprague-Dawley rats weighing between 300 and 350 g which were approved by the Experimental Animal Center of Nanjing Medical University. The rats were divided into two groups at random: renovascular hypertensive rats and sham rats. The renovascular hypertensive was induced with two-kidney one-clip (2K1C) method. The sham rats underwent the same operative procedure excepet their renal arteries were not occluded. The final experiment was carried out 4 weeks after the 2K1C or sham surgery. In 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 stereotaxie instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The superoxide anions level in the PVN was measured with lucigenin-derived chemiluminescence method. The NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescent detection of superoxide anions using a TD-20/20n Luminometer. The expression of c-Src and c-Src activity was measured by Western blotting method.1. The effects of PVN microinjection of saline, 25%DMSO, c-Src inhibitor PP2 (0.001, 0.01, 0.1 and 1 nmol) and SU6656 (0.5 nmol) on the CSAR, baseline RSNA and MAP were determined in 7 groups of 2K1C rats and 7 groups of sham rats (n=6 for each).2. The effects of pretreatment with PVN microinjection of saline, 25%DMSO, PP2 (0.001, 0.01, 0.1 and 1 nmol) and SU6656 (0.5 nmol) on the CSAR, RSNA and MAP responses to PVN microinjection of Ang?(0.3 nmol) were investigated in 7 groups of 2K1C rats and 7 groups of sham rats (n=6 for each).3. The effects of PVN microinjection of PP2 (1 nmol) on superoxide anions level and NAD(P)H oxidase activity in the PVN were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).4. The effects of pretreatment with PVN microinjection of PP2 (1 nmol) on superoxide anions level and NAD(P)H oxidase activity responses to PVN microinjection of Ang?(0.3 nmol) were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).5. The effects of pretreatment with PVN microinjection of PP2 (1 nmol) on superoxide anions level and NAD(P)H oxidase activity responses to epicardial application of capsaicin (1 nmol) were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).6. The effects of microinjection of saline and Ang?(0.3 nmol) on the expression of c-Src and c-Src activity in the PVN were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).7. The effects of epicardial application of saline and capsaicin (1 nmol) on the expression of c-Src and c-Src activity in the PVN were determined in 2 groups of 2K1C rats and 2 groups of sham rats (n=6 for each).1. Microinjection of c-Src inhibitor PP2 or SU6656 into the PVN significantly decreased the basal RSNA and MAP in both 2K1C rats and sham rats. The decreased RSNA effect in 2K1C rats was more obvious.2. The CSAR in 2K1C rats was enhanced. Microinjection of PP2 or SU6656 into the PVN inhibited the enhanced CSAR. The highest dose of PP2 abolished the CSAR.3. Pretreatment with microinjection of PP2 or SU6656 into the PVN attenuated the enhanced effects of Ang?on the basal RSNA and MAP in both 2K1C rats and sham rats. The highest dose of PP2 abolished the enhanced effects of Ang?in the PVN.4. Microinjection of Ang?into the PVN enhanced the CSAR, especially in 2K1C rats. Pretreatment with microinjection of PP2 or SU6656 into the PVN attenuated the enhanced effects of Ang?on the CSAR. The highest dose of PP2 abolished the enhanced effects of Ang?in the PVN.5. The c-Src activity,the NAD(P)H oxidase activity and superoxide anions level in the PVN were increased in 2K1C rats compared with sham rats,while the expression of c-Src had no significant difference between 2K1C rats and sham rats. Microinjection of the highest dose of PP2 into PVN significantly decreased superoxide anions level and the NAD(P)H oxidase activity in the PVN in 2K1C rats.6. Microinjection of Ang?(0.3 nmol) into the PVN also significantly increased the c-Src activity,the NAD(P)H oxidase activity and superoxide anions level in the PVN in both sham rats and 2K1C rats but had no significant effect on the expression of c-Src. The effects of Ang?in 2K1C rats were larger than those in sham rats. Pretreatment with microinjection of the highest dose of PP2 into PVN abolished these increased effects.7. Epicardial application of capsaicin (1 nmol) increased the c-Src activity,the NAD(P)H oxidase activity and superoxide anions level in the PVN in both sham rats and 2K1C rats but had no significant effect on the expression of c-Src, furthermore the increased degree in 2K1C rats was greater than that in sham rats. Pretreatment with microinjection of the highest dose of PP2 into PVN abolished the effects of epicardial application of capsaicin.1. Increased c-Src activity in the PVN is involved in the enhanced CSAR in hypertension.2. C-Src in the PVN modulates the effects of Ang?in the PVN in 2K1C hypertension.3. C-Src modulates CSAR and the effect of Ang?in the PVN through activating NAD(P)H oxidase and inducing ROS generation.