Increased (pro)renin receptor expression in the subfornical organ of hypertensive humans.

The central nervous system plays an important role in essential hypertension in humans and in animal models of hypertension through modulation of sympathetic activity and Na+ and body fluid homeostasis. Data from animal models of hypertension suggest that the renin-angiotensin system in the subfornical organ (SFO) of the brain is critical for hypertension development. We recently reported that the brain (pro)renin receptor (PRR) is a novel component of the brain renin-angiotensin system and could be a key initiator of the pathogenesis of hypertension. Here, we examined the expression level and cellular distribution of PRR in the SFO of postmortem human brains to assess its association with the pathogenesis of human hypertension. Postmortem SFO tissues were collected from hypertensive and normotensive human subjects. Immunolabeling for the PRR and a retrospective analysis of clinical data were performed. We found that human PRR was prominently expressed in most neurons and microglia, but not in astrocytes, in the SFO. Importantly, PRR levels in the SFO were elevated in hypertensive subjects. Moreover, PRR immunoreactivity was significantly correlated with systolic blood pressure but not body weight, age, or diastolic blood pressure. Interestingly, this correlation was independent of antihypertensive drug therapy. Our data indicate that PRR in the SFO may be a key molecular player in the pathogenesis of human hypertension and, as such, could be an important focus of efforts to understand the neurogenic origin of hypertension. NEW & NOTEWORTHY This study provides evidence that, in the subfornical organ of the human brain, the (pro)renin receptor is expressed in neurons and microglia cells but not in astrocytes. More importantly, (pro)renin receptor immunoreactivity in the subfornical organ is increased in hypertensive humans and is significantly correlated with systolic blood pressure.

Brain ACE2 overexpression reduces DOCA-salt hypertension independently of endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress.

Activation of the renin-angiotensin system, specifically in the subfornical organ is sufficient to induce fluid intake.

Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

Angiotensin-converting enzyme 2 overexpression in the subfornical organ prevents the angiotensin II-mediated pressor and drinking responses and is associated with angiotensin II type 1 receptor downregulation.

We recently reported the presence of angiotensin-converting enzyme (ACE)2 in brain regions controlling cardiovascular function; however, the role of ACE2 in blood pressure regulation remains unclear because of the lack of specific tools to investigate its function. We hypothesized that ACE2 could play a pivotal role in the central regulation of cardiovascular function by regulating other renin-angiotensin system components. To test this hypothesis, we generated an adenovirus expressing the human ACE2 cDNA upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-hACE2-eGFP). In vitro characterization shows that neuronal cells infected with Ad-hACE2-eGFP (10 to 100 multiplicities of infection), but not Ad-eGFP (100 multiplicities of infection), exhibit dose-dependent ACE2 expression and activity. In addition, an active secreted form was detected in the conditioned medium. In vivo, Ad-hACE2-eGFP infection (2x10(6) plaque-forming units intracerebroventricularly) produced time-dependent expression and activity (with a peak at 7 days) in the mouse subfornical organ. More importantly, 7 days after virus infection, the pressor response to angiotensin (Ang) II (200 pmol intracerebroventricularly) was significantly reduced in Ad-hACE2-eGFP-treated mice compared with controls. Furthermore, subfornical organ-targeted ACE2 overexpression dramatically reduced the Ang II-mediated drinking response. Interestingly, ACE2 overexpression was associated with downregulation of the Ang II type 1 receptor expression both in vitro and in vivo. These data suggest that ACE2 overexpression in the subfornical organ impairs Ang II-mediated pressor and drinking responses at least by inhibiting the Ang II type 1 receptor expression. Taken together, our results show that ACE2 plays a pivotal role in the central regulation of blood pressure and volume homeostasis, offering a new target for the treatment of hypertension and other cardiovascular diseases.

Protective actions of estrogen on angiotensin II-induced hypertension: role of central nitric oxide.

The present study tested the hypotheses that 1) nitric oxide (NO) is involved in attenuated responses to ANG II in female mice, and 2) there is differential expression of neuronal NO synthase (nNOS) in the subfornical organ (SFO) and paraventricular nucleus (PVN) in response to systemic infusions of ANG II in males vs. females. Aortic blood pressure (BP) was measured in conscious mice with telemetry implants. N(G)-nitro-l-arginine methyl ester (l-NAME; 100 microg x kg(.-1)day(-1)), an inhibitor of NOS, was administrated into the lateral cerebral ventricle for 14 days before and during ANG II pump implantation. Central infusion of l-NAME augmented the pressor effects of systemic ANG II in females (Delta21.5 + or - 2.2 vs. Delta9.2 + or - 1.5 mmHg) but not in males (Delta29.4 + or - 2.5 vs. Delta30.1 + or - 2.5 mmHg). Central administration of N(5)-(1-imino-3-butenyl)-l-ornithine (l-VNIO), a selective nNOS inhibitor, also significantly potentiated the increase in BP induced by ANG II in females (Delta17.5 + or - 3.2 vs. Delta9.2 + or - 1.5 mmHg). In gonadectomized mice, central l-NAME infusion did not affect the pressor response to ANG II in either males or females. Ganglionic blockade after ANG II infusion resulted in a greater reduction in BP in central l-NAME- or l-VNIO-treated females compared with control females. Western blot analysis of nNOS protein expression indicated that levels were approximately 12-fold higher in both the SFO and PVN of intact females compared with those in intact males. Seven days of ANG II treatment resulted in a further increase in nNOS protein expression only in intact females (PVN, to approximately 51-fold). Immunohistochemical studies revealed colocalization of nNOS and estrogen receptors in the SFO and PVN. These results suggest that NO attenuates the increase in BP induced by ANG II through reduced sympathetic outflow in females and that increased nNOS protein expression associated with the presence of female sex hormones plays a protective role against ANG II-induced hypertension in female mice.

Angiotensin II-Induced Hypertension Is Attenuated by Overexpressing Copper/Zinc Superoxide Dismutase in the Brain Organum Vasculosum of the Lamina Terminalis.

Angiotensin II (AngII) can access the brain via circumventricular organs (CVOs), including the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), to modulate blood pressure. Previous studies have demonstrated a role for both the SFO and OVLT in the hypertensive response to chronic AngII, yet it is unclear which intracellular signaling pathways are involved in this response. Overexpression of copper/zinc superoxide dismutase (CuZnSOD) in the SFO has been shown to attenuate the chronic hypertensive effects of AngII. Presently, we tested the hypothesis that elevated levels of superoxide (O2 (∙-)) in the OVLT contribute to the hypertensive effects of AngII. To facilitate overexpression of superoxide dismutase, adenoviral vectors encoding human CuZnSOD or control adenovirus (AdEmpty) were injected directly into the OVLT of rats. Following 3 days of control saline infusion, rats were intravenously infused with AngII (10 ng/kg/min) for ten days. Blood pressure increased 33 ± 8 mmHg in AdEmpty rats (n = 6), while rats overexpressing CuZnSOD (n = 8) in the OVLT demonstrated a blood pressure increase of only 18 ± 5 mmHg after 10 days of AngII infusion. These results support the hypothesis that overproduction of O2 (∙-) in the OVLT plays an important role in the development of chronic AngII-dependent hypertension.

Subcellular localization of nitric oxide synthase in the cerebral ventricular system, subfornical organ, area postrema, and blood vessels of the rat brain.

The distribution of neuronal nitric oxide synthase (nNOS) has been studied in the more rostral portion of the lateral ventricle, subfornical organ, area postrema and blood vessels of the rat central nervous system. nNOS was located by means of a specific polyclonal antibody, by using light and electron microscopy. Light microscopy showed immunoreactive varicose nerve fibers and terminal boutons-like structures in the lateral ventricle, positioned in supra- and subependimal areas. The spatial relationships between immunoreactive neuronal processes and the wall of the intracerebral blood vessels were studied. Electron microscopy showed numerous nerve fibers in the wall of the lateral ventricle; many were nNos-immunoreactive and established very close contact with ependymal cells. Immunoreactive neurons and processes were found in the subependymal plate of the ventricular wall, the subfornical organ, the area postrema, and the circularis nucleus of the hypothalamus. In these last three areas, the immunoreactive neurons were found close to the perivascular space of fenestrated and nonfenestrated blood vessels. The nNOS immunoreactivity was localized to the endoplasmic reticulum, cisterns, ribosomes, neurotubules, and in the inner part of the external membrane. In the terminal boutons, the reaction product was found surrounding the vesicle membranes. This distribution showed nNOS as a predominantly membrane-bound protein. The nitrergic nerve fibers present in the wall of the ventricular system might regulate metabolic functions as well as neurotransmission in the subfornical organ, area postrema and circularis nucleus of the hypothalamus.

Induction of pro-inflammatory cytokine mRNAs in the brain after peripheral injection of subseptic doses of lipopolysaccharide in the rat.

Although it is generally accepted that pro-inflammatory cytokines produced by cells of the central nervous system play important roles in the communication between the central nervous system and the immune system during sepsis, it is not clear whether these cytokines are produced in the brain under subseptic conditions. In this study, we used in situ hybridization to examine the mRNA expression of the pro-inflammatory cytokines IL-1beta and TNFalpha in the brains of rats 2 and 12 h after they were challenged by peripheral injections of lipopolysaccharide (LPS) ranging from 0.01 to 1000 microg/kg. Unlike septic doses of LPS (> 500 microg/kg), which induce global expression of pro-inflammatory cytokines in the brain, subseptic doses of LPS (0.01-10 microg/kg) induced IL-1beta and TNFalpha mRNA expression only in the choroid plexus, the circumventricular organs, and meninges. The expression of the cytokine-responsive immediate early gene I kappaB alpha was induced in the brain after doses of LPS as low as 0.1 microg/kg. I kappaB alpha mRNA expression was confined to sites where IL-1beta and TNFalpha were expressed. These results indicate that the induction and action of pro-inflammatory cytokines during subseptic infection occur at the blood-brain barrier and at circumventricular organs, which may be sites for elaboration of signal molecules that communicate peripheral immune status to the brain.

Aminopeptidases in the circumventricular organs of the mouse brain: a histochemical study.

The localization of four membrane-bound aminopeptidases--aminopeptidase A, aminopeptidase M, dipeptidylpeptidase IV, and gamma-glutamyl transpeptidase--known as characteristic enzymes of the blood-brain barrier was studied in the microvasculature of some circumventricular organs of the mouse brain (subfornical organ, area postrema, choroid plexus, and neurohypophysis). Enzyme activities were demonstrated histochemically in chloroform-acetone-pretreated cryostat sections applying an azo-coupling method. Reactions were evaluated using light microscopy and end-point microdensitometry. The results revealed differences in microvascular enzyme pattern between circumventricular organs and regions having a blood-brain barrier. Moreover, the cytochemical picture of the circumventricular organs themselves was not uniform. Dipeptidylpeptidase IV reaction showed a strongly reduced activity in the microvessels of all studied circumventricular organs. On the other hand, aminopeptidase M seemed to be present in both the leaky and the tight capillaries. Only a low activity of aminopeptidase A was found in parts of the choroid endothelium and the subfornical organ microvasculature. gamma-Glutamyl transpeptidase could neither be detected in the capillary part of the choroid plexus nor in the neurohypophysis. We are led to conclude that at least dipeptidylpeptidase IV might be involved in special mechanisms of the blood-brain barrier.

Angiotensin-converting enzyme in subfornical organ mediates captopril-induced drinking.

These experiments tested whether angiotensin-converting enzyme (ACE) located within the subfornical organ (SFO) participates in the generation of water intake during peripheral ACE blockade with captopril (CAP). Lesions of the SFO virtually abolished drinking in response to intraperitoneal CAP injection. Intracranially injected CAP suppressed drinking induced by intraperitoneal CAP more completely with direct SFO injection compared with intraventricular or control tissue injections. This central captopril treatment did not alter the drinking response to subcutaneous hypertonic saline. Intraventricular injections of the angiotensin II (ANG II) receptor blocker sarile reduced drinking during oral captopril treatment in rats rehydrating from water deprivation. The results indicate that (a) the SFO mediates drinking caused by peripheral ACE inhibition; (b) the ACE located within the SFO may locally convert ANG I to ANG II, which then stimulates thirst; and (c) central ANG II receptors mediate thirst caused by peripheral ACE inhibition.

Quantitative distribution of angiotensin-converting enzyme (kininase II) in discrete areas of the rat brain by autoradiography with computerized microdensitometry.

We report the localization of angiotensin-converting enzyme (kininase II, EC 3.4.15.1) in discrete nuclei and areas of the rat brain by a quantitative autoradiographic technique using image processing coupled to computerized microdensitometry, after incubation of brain sections with the specific converting enzyme inhibitor [125I]351A. High angiotensin-converting enzyme levels are present in circumventricular organs (organon subfornicalis and area postrema), the choroid plexus, and extrapyramidal areas (nucleus caudatus, globus pallidus and substantia nigra) with intermediate levels in selected hypothalamic, septal, habenular and brainstem nuclei. Our results support the idea that angiotensin II could be formed in specific brain areas, both outside and inside the blood-brain barrier. In other brain structures, such as the extrapyramidal areas, kininase II could be involved in the processing or metabolism of other brain peptides.

Tyrosine hydroxylase-immunoreactive projections from the caudal ventrolateral medulla to the subfornical organ in the rat.

The subfornical organ (SFO) is known to be innervated by noradrenergic fibers. One possible origin of these fibers, which carry peripheral baroreceptor information to enhance the activity of SFO neurons, is the nucleus tractus solitarius (NTS). To investigate possible sites of origin of the catecholaminergic projections to the SFO, a retrograde tracing method was combined with immunohistochemistry in the rat. Stereotaxical injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase--colloidal gold complex, into the SFO from the dorsal aspect revealed retrogradely labeled neurons in several catecholaminergic cell groups. A substantial number of retrogradely labeled neurons showing tyrosine hydroxylase (TH) immunoreactivity were found in the NTS and ventrolateral medulla (VLM) at levels caudal to the obex and in the locus coeruleus, while retrogradely labeled neurons without TH immunoreactivity were found in the VLM at levels rostral to the obex and in the nucleus prepositus hypoglossi. When the tracer was injected into the structures dorsal to the SFO, including the triangular septal nucleus, the frequency of retrogradely labeled neurons in the NTS and VLM at the caudal level was very low. These findings indicate the existence of catecholaminergic projections from the VLM (probably A1) to the SFO, in addition to the noradrenergic projections from the NTS previously reported.

Immunohistochemical localization of catechol-O-methyltransferase in circumventricular organs of the rat: potential variations in the blood-brain barrier to native catechols.

Catechol-O-methyltransferase (COMT) was localized in the organum vasculosum of the lamina terminals, subfornical organ, subcommissural organ and area postrema of rat brain using an indirect immunofluorescence technique. COMT immunofluorescence was apparent in neuroglia within the organum vasculosum and was most intense in the ependyma between this structure and the optic recess of the third ventricle. In both the subfornical organ and the area postrema, COMT was localized in a neuroglial network, but was noticeably absent in the ependymal layer. COMT immunofluorescence in the ependyma of the subcommissural organ was continuous with the more intense immunofluorescence of the cuboidal ependyma of the third ventricle. Each of the circumventricular organs studied, with the exception of the subcommissural organ, lies outside the blood-brain barrier. However, the unique pattern of COMT immunofluorescence in the area postrema and the subfornical organ suggests that these two structures, of all circumventricular organs, are most likely to permit the entry of peripherally circulating catechols to the cerebrospinal fluid.

Angiotensin-converting enzyme in discrete areas of the rat forebrain and pituitary gland.

With the use of a sensitive radioisotopic method we have examined the activity of the angiotensin-converting enzyme (ACE, E.C. 3.4.15.1) in specific nuclei of the rat forebrain and in the anterior, intermediate and posterior lobes of the pituitary gland of the rat. We reported that ACE activity is heterogeneously distributed in the rat forebrain, with a 200-fold difference between the lowest and the highest values. Highest enzyme activities were found in the subfornical organ and in the posterior lobe of the pituitary gland. High ACE activity was also detected in the intermediate and anterior lobes of the pituitary gland, the caudate nucleus, and the medial habenular nucleus. Substantial activity also existed in the globus pallidus, the median eminence, the supraoptic and paraventricular nuclei, the lateral habenular nucleus and the organon vasculosum laminae terminalis. Our results demonstrate that one of the components of the renin-angiotensin system, the angiotensin-converting enzyme, is highly localized to a few discrete brain structures and the pituitary gland. These findings suggest that angiotensin II could be formed locally in some of these structures, supporting previous immunohistochemical data.

Primary culture of circumventricular organs from the rat brain lamina terminalis.

A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH-diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes.

Angiotensin-converting enzyme is present in the subfornical organ and other circumventricular organs of the rat.

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) activity was measured by a radiochemical assay in isolated circumventricular organs of the rat. ACE activity was unevenly distributed, with a 100-fold difference between the lowest (subcommisural) and the highest (subfornical organ) activities. Our results suggest that angiotensin II could be locally formed in circumventricular organs and especially in the subfornical organ. The high angiotensin-converting enzyme activity in the subfornical organ could indicate a physiological role of endogenous angiotensin II in this structure.

Central inhibition of nitric oxide synthase attenuates water intake but does not alter enhanced glucose utilization in the hypothalamo-neurohypophysial system of dehydrated rats.

I.c.v. administration of a nitric oxide (NO) synthase inhibitor (NG-monomethyl-L-arginine, NMMA, 500 micrograms/5 microliters) to conscious rats deprived of water for 24 h attenuated drinking and decreased glucose utilization in the subfornical organ and median preoptic nucleus. NMMA did not alter the enhanced glucose utilization in the hypothalamo-neurohypophysial system (HNS) of dehydrated rats, although it has been shown to increase, selectively, oxytocin (OT) secretion [18]. This suggests that NO may act in the neural lobe to inhibit OT secretion and promote the preferential release of vasopressin during dehydration. This effect is similar to the blockade of endogenous opiate receptors by naloxone.