Mineralocorticoid and angiotensin II type 1 receptors in the subfornical organ mediate angiotensin II - induced hypothalamic reactive oxygen species and hypertension.

Activation of angiotensinergic pathways by central aldosterone (Aldo)-mineralocorticoid receptor (MR) pathway plays a critical role in angiotensin II (Ang II)-induced hypertension. The subfornical organ (SFO) contains both MR and angiotensin II type 1 receptors (AT1R) and can relay the signals of circulating Ang II to downstream nuclei such as the paraventricular nucleus (PVN), supraoptic nucleus (SON) and rostral ventrolateral medulla (RVLM). In Wistar rats, subcutaneous (sc) infusion of Ang II at 500ng/min/kg for 1 or 2weeks increased reactive oxygen species (ROS) as measured by dihydroethidium (DHE) staining in a nucleus - specific pattern. Intra-SFO infusion of AAV-MR- or AT1aR-siRNA prevented the Ang II-induced increase in AT1R mRNA expression in the SFO and decreased MR mRNA. Both MR- and AT1aR-siRNA prevented increases in ROS in the PVN and RVLM. MR- but not AT1aR-siRNA in the SFO prevented the Ang II-induced ROS in the SON. Both MR- and AT1aR-siRNA in the SFO prevented most of the Ang II-induced hypertension as assessed by telemetry. These results indicate that Aldo-MR signaling in the SFO is needed for the activation of Ang II-AT1R-ROS signaling from the SFO to the PVN and RVLM. Activation of Aldo-MR signaling from the SFO to the SON may enhance AT1R dependent activation of pre-sympathetic neurons in the PVN.

Possible role of brain salt-inducible kinase 1 in responses to central sodium in Dahl rats.

In Dahl salt-sensitive (S) rats, Na(+) entry into the cerebrospinal fluid (CSF) and sympathoexcitatory and pressor responses to CSF Na(+) are enhanced. Salt-inducible kinase 1 (SIK1) increases Na(+)/K(+)-ATPase activity in kidney cells. We tested the possible role of SIK1 in regulation of CSF [Na(+)] and responses to Na(+) in the brain. SIK1 protein and activity were lower in hypothalamic tissue of Dahl S (SS/Mcw) compared with salt-resistant SS.BN13 rats. Intracerebroventricular infusion of the protein kinase inhibitor staurosporine at 25 ng/day, to inhibit SIK1 further increased mean arterial pressure (MAP) and HR but did not affect the increase in CSF [Na(+)] or hypothalamic aldosterone in Dahl S on a high-salt diet. Intracerebroventricular infusion of Na(+)-rich artificial CSF caused significantly larger increases in renal sympathetic nerve activity, MAP, and HR in Dahl S vs. SS.BN13 or Wistar rats on a normal-salt diet. Intracerebroventricular injection of 5 ng staurosporine enhanced these responses, but the enhancement in Dahl S rats was only one-third that in SS.BN13 and Wistar rats. Staurosporine had no effect on MAP and HR responses to intracerebroventricular ANG II or carbachol, whereas the specific protein kinase C inhibitor GF109203X inhibited pressor responses to intracerebroventricular Na(+)-rich artificial CSF or ANG II. These results suggest that the SIK1-Na(+)/K(+)-ATPase network in neurons acts to attenuate sympathoexcitatory and pressor responses to increases in brain [Na(+)]. The lower hypothalamic SIK1 activity and smaller effect of staurosporine in Dahl S rats suggest that impaired activation of neuronal SIK1 by Na(+) may contribute to their enhanced central responses to sodium.

Central neuronal activation and pressor responses induced by circulating ANG II: role of the brain aldosterone-"ouabain" pathway.

An increase in plasma ANG II causes neuronal activation in hypothalamic nuclei and a slow pressor response, presumably by increasing sympathetic drive. We evaluated whether the activation of a neuromodulatory pathway, involving aldosterone and "ouabain," is involved in these responses. In Wistar rats, the subcutaneous infusion of ANG II at 150 and 500 ng x kg(-1) x min(-1) gradually increased blood pressure up to 60 mmHg at the highest dose. ANG II at 500 ng x kg(-1) x min(-1) increased plasma ANG II by 4-fold, plasma aldosterone by 25-fold, and hypothalamic aldosterone by 3-fold. The intracerebroventricular infusion of an aldosterone synthase (AS) inhibitor prevented the ANG II-induced increase in hypothalamic aldosterone without affecting the increase in plasma aldosterone. Neuronal activity, as assessed by Fra-like immunoreactivity, increased transiently in the subfornical organ (SFO) but progressively in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). The central infusion of the AS inhibitor or a mineralocorticoid receptor blocker markedly attenuated the ANG II-induced neuronal activation in the PVN but not in the SON. Pressor responses to ANG II at 150 ng x kg(-1) x min(-1) were abolished by an intracerebroventricular infusion of the AS inhibitor. Pressor responses to ANG II at 500 ng x kg(-1) x min(-1) were attenuated by the central infusion of the AS inhibitor or the mineralocorticoid receptor blocker by 70-80% and by Digibind (to bind "ouabain") by 50%. These results suggest a novel central nervous system mechanism for the ANG II-induced slow pressor response, i.e., circulating ANG II activates the SFO, leading to the direct activation of the PVN and SON, and, in addition, via aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Effects of central sodium on epithelial sodium channels in rat brain.

We evaluated the effects of intracerebroventricular (icv) infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial Na(+) channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11beta-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF Na(+) concentration ([Na(+)]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. Na(+)-rich aCSF increased beta-ENaC mRNA and immunoreactivity in the choroid plexus and increased alpha- and beta-ENaC immunoreactivities in the ependyma. Na(+)-rich aCSF increased alpha- and beta-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in beta-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, Na(+)-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [Na(+)] in the control, but not Na(+)-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [Na(+)] by Na(+)-rich aCSF. These results suggest that CSF Na(+) upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of Na(+) homeostasis in the brain.

Role of central nervous system aldosterone synthase and mineralocorticoid receptors in salt-induced hypertension in Dahl salt-sensitive rats.

In Dahl salt-sensitive (S) rats, high salt intake increases cerebrospinal fluid (CSF) Na(+) concentration ([Na(+)]) and blood pressure (BP). Intracerebroventricular (ICV) infusion of a mineralocorticoid receptor (MR) blocker prevents the hypertension. To assess the role of aldosterone locally produced in the brain, we evaluated the effects of chronic central blockade with the aldosterone synthase inhibitor FAD286 and the MR blocker spironolactone on changes in aldosterone and corticosterone content in the hypothalamus and the increase in CSF [Na(+)] and hypertension induced by high salt intake in Dahl S rats. After 4 wk of high salt intake, plasma aldosterone and corticosterone were not changed, but hypothalamic aldosterone increased by approximately 35% and corticosterone tended to increase in Dahl S rats, whereas both steroids decreased by approximately 65% in Dahl salt-resistant rats. In Dahl S rats fed the high-salt diet, ICV infusion of FAD286 or spironolactone did not affect the increase in CSF [Na(+)]. ICV infusion of FAD286 prevented the increase in hypothalamic aldosterone and 30 mmHg of the 50-mmHg BP increase induced by high salt intake. ICV infusion of spironolactone fully prevented the salt-induced hypertension. These results suggest that, in Dahl S rats, high salt intake increases aldosterone synthesis in the hypothalamus and aldosterone acts as the main MR agonist activating central pathways contributing to salt-induced hypertension.

Central infusion of aldosterone synthase inhibitor attenuates left ventricular dysfunction and remodelling in rats after myocardial infarction.

AIMS: Blockade of mineralocorticoid receptors in the central nervous system (CNS) prevents sympathetic hyperactivity and improves left ventricle (LV) function in rats post-myocardial infarction (MI). We examined whether aldosterone produced locally in the brain may contribute to the activation of mineralocorticoid receptors in the CNS. METHODS AND RESULTS: Two days after coronary artery ligation, Wistar rats received an intra-cerebroventricular (icv) infusion via osmotic mini-pumps of the aldosterone synthase inhibitor FAD286 at 100 microg/kg/day or vehicle for 4 weeks. LV function was assessed by echocardiography at 2 and 4 weeks, and by Millar catheter at 4 weeks. At 4 weeks post-MI, aldosterone in the hippocampus was increased by 70% and tended to increase in the hypothalamus by 20%. These increases were prevented by FAD286. Across groups, aldosterone in the hippocampus and hypothalamus showed a high correlation. There were no differences in brain corticosterone levels. Compared to sham rats, at both 2 and 4 weeks post-MI rats treated with vehicle showed increased LV dimensions and decreased LV ejection fraction. Icv infusion of FAD286 attenuated these changes in LV dimensions and ejection fraction by approximately 30%. At 4 weeks post-MI, LV peak systolic pressure (LVPSP) and dP/dt(max/min) were decreased and LV end-diastolic pressure (LVEDP) was increased. In rats treated with icv FAD286, LVPSP and dP/dt(min) remained normal and LVEDP and dP/dt(max) were markedly improved. Post-MI increases in cardiac fibrosis and cardiomyocyte diameter were substantially attenuated by icv FAD286. CONCLUSION: These data suggest that aldosterone produced locally in the brain acts as the main agonist of mineralocorticoid receptors in the CNS and contributes substantially to the progressive heart failure post MI.

Brain Na+,K+-ATPase isozyme activity and protein expression in ouabain-induced hypertension.

In normotensive rats, chronic infusion of exogenous ouabain causes hypertension involving central mechanisms. To determine whether ouabain-induced hypertension is associated with specific changes in brain Na+,K+-ATPase activity and expression, we assessed brain Na+,K+-ATPase isozyme activity and protein expression in rats treated with ouabain (50 microg/day s.c. or 10 microg/day i.c.v. for 14 days). Resting mean arterial pressure (MAP) was higher in s.c.- and i.c.v.-ouabain-treated animals vs. control (124+/-2 vs. 105+/-2 and 130+/-2 vs. 109+/-2, respectively, p<0.01). Ouabain infused s.c. or i.c.v. for 14 days had no effect on Na+,K+-ATPase isozyme activity in hypothalamic, pontine/medullary or cortical microsomes. However, the percent increase in total Na+,K+-ATPase activity produced in vitro by antibody Fab fragments that bind ouabain with high affinity (Digibind) was two-fold greater in s.c.- and i.c.v.-ouabain-treated rats vs. control, but only in hypothalamic microsomes. Thus, ouabain infused s.c. or i.c.v. does appear to directly inhibit Na+,K+-ATPase activity in the hypothalamus. On the other hand, in the hypothalamus, s.c.- and i.c.v.-ouabain infusions tended to increase alpha3 (by 30-44%), but had no effect on alpha1 or alpha2 Na+,K+-ATPase isozyme protein expression. In addition, ouabain was found to partially dissociate from the Na+,K+-ATPase enzyme following sample processing. Thus, the inability to detect a decrease in enzyme activity in the hypothalamus in response to ouabain may be due, in part, to an increase in enzyme expression and the dissociation of ouabain during sample processing.

Prevention of sympathetic and cardiac dysfunction after myocardial infarction in transgenic rats deficient in brain angiotensinogen.

To provide evidence for the role of angiotensin II locally produced in the brain in the development of sympathetic hyperactivity and heart failure after myocardial infarction (MI), transgenic rats (TGR) were used, which express an antisense RNA against angiotensinogen. In TGR and control Sprague-Dawley (SD) rats, an MI was induced by acute coronary artery ligation. At 8 weeks after MI, MI sizes were similar in TGR and SD rats. In the groups with MI > or =25% of left ventricle (LV), LV peak systolic pressure decreased in SD rats but not in TGR. LV end-diastolic pressure increased substantially more in SD-MI than TGR-MI rats (from 2+/-1 to 15+/-2 mm Hg, and 2+/-1 to 8+/-1 mm Hg, respectively; P<0.05). LV dP/dtmax decreased from approximately 5400 to 3573+/-187 in SD-MI rats, but only to 4353+/-180 mm Hg/sec in TGR-MI (P<0.05). LV pressure volume curves in vitro showed a marked shift to the right in SD-MI rats. This shift was significantly attenuated by -70% in TGR versus SD rats with MI. Both RV weight and interstitial fibrosis in the LV increased clearly in the SD-MI rats, but not or significantly less in the TGR-MI rats. In SD-MI rats, arterial baroreflex control of heart rate and renal sympathetic nerve activity was markedly impaired but was not affected in the TGR-MI. Plasma angiotensin II levels tended to be higher in SD versus TGR rats, both in sham and MI-groups. This study provides the major new finding that in rats after MI, angiotensin II locally produced in the brain plays a dominant role in the development of LV dysfunction after MI, possibly through its effects on sympathetic function and on circulatory/cardiac renin-angiotensin system.