The angiotensin II pressor system of the rat forebrain

An anterior hypothalamic knife cut that leaves intact two central sites of action of angiotension II produces the same deficits in the pressor responses to angiotensin II that have been attributed to destruction of two circumventricular organs (the subfornical organ and the organum vasculosum of the lamina terminalis). The central pressor actions of angiotensin II are necessary for the full expression of renin-dependent renal hypertension. The anterior hypothalamic knife cut attenuates renin-dependent aortic ligation hypertension. It has been shown that electrolytic destruction more anterior to this knife cut, in the anteroventral 3rd ventricle region, also attenuates two forms of renin-dependent hypertension, aortic ligation hypertension and two-kidney Goldblatt hypertension in the rat. Electrolytic lesions in the subfornical organ also reduce renin-dependent, two-kidney Goldblatt hypertension. These data are consistent with the hypothesis that a common efferent system from the organum vasculosum of the lamina terminalis and subfornical organ mediates the central pressor response to angiotensin II and is involved in the development of renin-dependent hypertension. A model is proposed for the circuitry in the rat forebrain that is involved in the pressor response to angiotensin II.     

Effect of dopamine injection into the anteroventral third ventricular region and the paraventricular nucleus on vasopressin secretion in conscious rats.

To investigate the role of dopamine receptors situated in the paraventricular nucleus and the anteroventral third ventricular region in regulating vasopressin release, responses of plasma AVP and its controlling factors to injections of dopamine into these regions and the lateral cerebral ventricle were examined in conscious rats. The injections of 156 nmol (30 micrograms) dopamine into the cerebral ventricle produced transient rises in plasma AVP 5 min later. When the dose of dopamine was reduced to 26 nmol (5 micrograms), the increase in plasma AVP was not provoked any more. However, injections of 26 nmol dopamine into the paraventricular nucleus greatly augmented plasma AVP 5 and 15 min later. This dose of dopamine was without effect on plasma AVP when injected into the anteroventral third ventricular region, including the organum vasculosum lamina terminalis, median preoptic nucleus, medial preoptic area and the periventricular preoptic nucleus. These dopamine administrations in the cerebral ventricle, paraventricular nucleus and the anteroventral third ventricular region did not significantly change AVP-controlling factors such as plasma osmolality, sodium and arterial pressure. On the basis of these results, we conclude that dopamine receptors in the paraventricular nucleus may function to facilitate AVP secretion, whereas those in the anteroventral third ventricular region may not play an important role in the regulation of AVP release.     

Differential angiotensin-converting enzyme inhibition in brain after oral administration of perindopril demonstrated by quantitative in vitro autoradiography

To help elucidate potential sites for the central actions of a new angiotensin-converting enzyme (ACE) inhibitor, perindopril, ACE levels were measured in the brain of Sprague-Dawley rats by quantitative in vitro autoradiography after administration of the drug. Following acute oral administration of 1 mg/kg perindopril, ACE in the two circumventricular organs, the subfornical organ and organum vasculosum of the lamina terminalis, was markedly inhibited and had only partially recovered after 24 h. The ACE inhibition in the circumventricular organs did not correlate with the inhibition of ACE in plasma but with that of pressor response to intravenous angiotensin I. No or little change in ACE was observed in other brain structures which are rich in the enzyme, including the choroid plexus and basal ganglia. However, large doses of perindopril (up to 16 mg/kg) did progressively inhibit ACE in all brain structures measured, including the basal ganglia. These findings fit with the deficient blood-brain barrier known to occur in the circumventricular organs. These regions are rich in ACE and angiotensin II receptors and exhibit physiological responses to angiotensin II with effects on fluid, electrolyte, and blood pressure homeostasis. Combined with current observations, the circumventricular organs are potential targets for the centrally mediated actions of ACE inhibitors.     

Atrial natriuretic factor: neuromodulator of the central nervous system regulation of blood pressure

Extensive examination in the mammalian brain for the presence of atrial natriuretic factors (ANF) has revealed both peptide and receptors specifically distributed throughout the central nervous system. High concentrations of ANF have been found in several hypothalamic nuclei, septal areas, the anteroventral third ventricular area (AV3V), and the median eminence, whereas moderate concentrations have been detected in the circumventricular organs and several brainstem nuclei. The receptors for ANF have been found in moderate to high concentrations in the olfactory lobe, AV3V region, and several circumventricular organs (subfornical organ, organum vasculosum of the lamina terminalis) as well as the nucleus tractus solitarius, median eminence, and choroid plexus. These findings suggest a role for ANF in modulating fluid and electrolyte balance, and blood pressure in this compartment, analogous to the proposed actions of this peptide hormone in the periphery. To determine whether ANF might function as a neuromodulator of blood pressure, we administered ANF via fourth ventricular injection into the brain of hypertensive (SHR) and normotensive rats (WKY). Atrial natriuretic factor caused a moderate and significant decrease in mean arterial blood pressure in both strains. The action of ANF appeared to be mediated by activating the central alpha 2-adrenergic nervous system, probably through the release of catecholamines. Further, a dependence on the secretion and action of an endogenous opioid was probably involved; heart rate was unaffected in these studies. Experiments from other laboratories indicate that central ANF may modulate the pressor effects of centrally acting angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating angiotensin II and dietary salt: Converging signals for neurogenic hypertension

Circulating angiotensin II (Ang II) combined with high salt intake increases sympathetic nerve activity (SNA) in some forms of hypertension. Ang II-induced increases in SNA are modest, delayed, and specific to certain vascular beds. The brain targets for circulating Ang II are neurons in the area postrema (AP), subfornical organ (SFO), and possibly other circumventricular organs. Ang II signaling is integrated with sodium-sensitive neurons in the SFO and/or organum vasculosum of the lamina terminalis (OVLT) and drives sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) via the paraventricular nucleus (PVN). It is likely that, over time, new patterns of gene expression emerge within neurons of the SFO-PVN-RVLM pathway that transform their signaling properties. This transformation is critical in maintaining increased SNA. Identification of a novel gene supporting this process may provide new targets for treatment of neurogenic hypertension.     

Short-term effect of the lesion of the organum vasculosum of the lamina terminalis on hypothalamic LH-RH and serum LH, FSH and prolactin in adult female rats.

The short-term effect of the mechanical lesion of the organum vasculosum of the lamina terminalis (OVLT) was investigated in 4-day cycling female rats. The lesions were performed on the 2nd day of diestrus, and the animals were killed by decapitation 30 h after the lesion. Serum LH, FSH and prolactin and hypothalamic LH-RH content of 3 different parts of the hypothalamus were determined with radioimmunoassay. OVLT lesion caused a significant increase in the LH-RH content of the mid-basal hypothalamus and in serum prolactin levels and a decrease in LH and FSH serum levels. The results support the view that the OVLT may play a role in the control of pituitary gonadotrophic hormone secretion.     

Localization of central angiotensin II receptors with [125I]-sar1, ile8-angiotensin II: periventricular sites of the anterior third ventricle

The radiolabeled angiotensin II (ANG II) antagonist, [N 125I]-sar1,ile8-ANG II, was used to study brain ANG II receptors by both homogenate binding and in vitro autoradiography. In homogenate preparations of the hypothalamus, thalamus, septum and midbrain (HTSM), [125I]-sar1,ile8-ANG II bound to a single class (Hill slope 0.84 +/- 0.05) of high affinity binding sites (KD 0.42 +/- 0.03 nM, Bmax 5.98 +/- fmol/mg protein). Competition for the [125I]-sar1,ile8-ANG II binding site in HTSM membranes demonstrated a rank order potency characteristic of binding to the ANG II receptor, with the unlabeled antagonist being slightly more potent than ANG II (Ki 0.22 +/- 0.03 vs 0.95 +/- 0.06 nM, respectively). Brain slices from the region of the rostral third ventricle were incubated with 0.5 nM[125I]-sar1,ile8-ANG II in the presence or absence of 1 microM ANG II and exposed to LKB Ultrofilm. Autoradiographic images of [125I]-sar1,ile8-ANG II binding revealed that structures situated within the anterior wall of the third ventricle, i.e. the lamina terminalis, were heavily labeled; including the subfornical organ, median preoptic nucleus and organum vasculosum laminae terminalis. These results show the utility of [125I]-sar1,ile8-ANG II as a probe to study brain ANG II receptors and provides pharmacological evidence for the rostral third ventricle as a possible site for central ANG II actions.     

The brain as an endocrine target for Peptide hormones.

Unlike circulating steroid hormones, which have a relatively unhindered passage into the central nervous system, blood-borne peptides are usually restricted by the blood-brain barrier. Some circulating peptides, such as angiotensin II, atrial natriuretic peptide and relaxin, influence central neural pathways subserving cardiovascular and body fluid homeostasis by acting on neurons in the subfornical organ, organum vasculosum of the lamina terminalis and area postrema, all of which lack a blood-brain barrier. There are some circulating peptides such as insulin and leptin that are transported from the bloodstream across cerebral blood vessel walls into sites in the hypothalamus that have appropriate neural connections to influence food intake and sympathetic control of brown fat.     

Angiotensin II receptor binding in the rat hypothalamus and circumventricular organs during dietary sodium deprivation

The effect of dietary sodium intake on angiotensin II (Ang II) receptor binding in the rat brain was studied using quantitative in vitro autoradiography. After 2 weeks of sodium deprivation, the peripheral angiotensin system was activated as shown by increased plasma renin activity (4-fold) and plasma aldosterone concentration (approximately 40-fold). At the same time, Ang II receptor binding in the adrenal glomerulosa zone increased by 40%. Frozen brain sections prepared from 12 male Sprague-Dawley rats (6 control, 6 sodium-deprived) were incubated with 125I[Sar1, Ile8] Ang II, exposed to X-ray film, and Ang II receptor binding in individual brain nuclei was quantitated by computerized densitometry. Ang II binding in the area postrema was significantly suppressed in the sodium-deprived rats (60% of control; p less than 0.05). No change was observed in the other circumventricular organs studied, the subfornical organ or organum vasculosum of the lamina terminalis. Ang II binding in the solitary tract nucleus was not affected by the dietary salt treatment. In the hypothalamic paraventricular nucleus, there was a small (9%) but significant (p less than 0.001) increase in Ang II receptor binding in the sodium-deprived group. However, no change was observed in the hypothalamic median preoptic or suprachiasmatic nuclei, areas with similarly high Ang II receptor binding. These results suggest that only a limited subset of brain Ang II receptors respond to sodium deprivation and do so in a region-specific manner. These results support evidence that the central angiotensin system may contribute to the regulation of fluid and electrolyte homeostasis.     

Angiotensin type 1 receptors in the subfornical organ mediate the drinking and hypothalamic-pituitary-adrenal response to systemic isoproterenol

Circulating angiotensin II (ANGII) elicits water intake and activates the hypothalamic-pituitary-adrenal (HPA) axis by stimulating angiotensin type 1 receptors (AT1Rs) within circumventricular organs. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) are circumventricular organs that express AT1Rs that bind blood-borne ANGII and stimulate integrative and effector regions of the brain. The goal of these studies was to determine the contribution of AT1Rs within the SFO and OVLT to the water intake and HPA response to increased circulating ANGII. Antisense oligonucleotides directed against the AT1R [AT1R antisense (AT1R AS)] were administered into the OVLT or SFO. Quantitative receptor autoradiography confirmed that AT1R AS decreased ANGII binding in the SFO and OVLT compared with the scrambled sequence control but did not affect AT1R binding in other nuclei. Subsequently, water intake, ACTH, and corticosterone (CORT) were assessed after administration of isoproterenol, a beta-adrenergic agonist that decreases blood pressure and elevates circulating ANGII. Delivery of AT1R AS into the SFO attenuated water intake, ACTH, and CORT after isoproterenol, whereas similar treatment in the OVLT had no effect. To determine the specificity of this blunted drinking and HPA response, the same parameters were measured after treatment with hypertonic saline, a stimulus that induces drinking independently of ANGII. Delivery of AT1R AS into the SFO or OVLT had no effect on water intake, ACTH, or CORT after hypertonic saline. The results imply that AT1R within the SFO mediate drinking and HPA responses to stimuli that increase circulating ANGII.     

Angiotensin II receptors in the ventral portion of the bed nucleus of the stria terminalis

In vitro receptor autoradiography, using the radiolabeled angiotensin II (Ang II) antagonist 125I-sar1,ile8 Ang II (125I-SI Ang II; 250 pM) in the absence or presence of 1 microM Ang II, was used to identify Ang II receptor binding sites in the preoptic-anterior hypothalamic (POAH) brain region of cycling female rats. A nucleus within this region, lateral to the organum vasculosum of the lamina terminalis and ventral to the anterior commissure, displayed a discrete locus of 125I-SI Ang II binding sites (385 fmol/g tissue). This nucleus, which corresponds to the area of the POAH from which Ang II is most effective at eliciting luteinizing hormone release, has been identified as the ventral portion of the bed nucleus of the stria terminalis (BSTV) by the rat brain atlas of Paxinos and Watson. The selective nonpeptidic Ang II alpha receptor antagonist Dup 753 completely inhibited the binding of 125I-SI Ang II to the BSTV and other hypothalamic nuclei, suggesting that these receptors are of the Ang II alpha subtype.     

Effects of delta-9-tetrahydrocannabinol on the hypothalamic-pituitary control of luteinizing hormone and follicle-stimulating hormone secretion in adult male rats

The main psychoactive component of marihuana, delta-9-tetrahydrocannabinol (THC) was injected into the 3rd cerebral ventricle. A single dose of THC (2 microliter of 10(-6) M) decreased serum LH temporarily but did not alter serum follicle-stimulating hormone (FSH) levels. The mediobasal hypothalamic (MBH) luteinizing hormone-releasing hormone (LHRH) content was elevated by 30 min after the injection. The elevation persisted for 1 h. Then, the LHRH content returned towards the preinjection level. In contrast, the LHRH in the organum vasculosum of the lamina terminalis did not change after a single dose of THC. The results indicate that THC alters pituitary LH release by inhibiting the release of LHRH which then increases in the MBH by continued synthesis or transport from rostral areas. In addition, the data support the existence of an FSH releasing factor, the release of which is not suppressed by this dose of THC. THC did not alter the release, storage or responsiveness to LHRH of cultured anterior pituitary cells, which further supports the view that its principal site of action is on the hypothalamus.     

Role of the organum vasculosum laminae terminalis in the control of gonadotrophin secretion in rats

The organum vasculosum laminae terminalis (OVLT) was destroyed by radiofrequency lesions in regularly cycling and in long-term ovariectomized adult rats. After OVLT lesion practically all cyclic females (16 out of 22) became dioestrous, as indicated by vaginal smears. At the time of killing these animals (8 days after the lesion) serum LH levels were undetectable, while serum FSH was as low as in cyclic animals in dioestrus. In the few OVLT-lesioned animals which exhibited some sort of oestrous cyclicity, serum LH showed a small subphysiological increase at pro-oestrus; this was not accompanied by a parallel increase in serum FSH and in these animals a delayed peak of FSH occurred on the day of oestrus. Ovariectomized rats bearing OVLT lesions had serum titres of Lh and FSH as high as those of ovariectomized control rats. It is suggested that the OVLT may play a role in the control of the cyclic release of gonadotrophins but is not involved in the tonic regulation of gonadotrophin secretion.     

Inhibition of tissue angiotensin converting enzyme. Quantitation by autoradiography

Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after administration of lisinopril, an ACE inhibitor. Tissue ACE was assessed by quantitative in vitro autoradiography using the ACE inhibitor [125I]351A, as a ligand, and serum ACE was measured by a fluorimetric method. Following oral administration of lisinopril (10 mg/kg), serum ACE activity was acutely reduced but recovered gradually over 24 hours. Four hours after lisinopril administration, ACE activity was markedly inhibited in kidney (11% of control level), adrenal (8%), duodenum (8%), and lung (33%; p less than 0.05). In contrast, ACE in testis was little altered by lisinopril (96%). In brain, ACE activity was markedly reduced 4 hours after lisinopril administration in the circumventricular organs, including the subfornical organ (16-22%) and organum vasculosum of the lamina terminalis (7%; p less than 0.05). In other areas of the brain, including the choroid plexus and caudate putamen, ACE activity was unchanged. Twenty-four hours after administration, ACE activity in peripheral tissues and the circumventricular organs of the brain had only partially recovered toward control levels, as it was still below 50% of control activity levels. These results establish that lisinopril has differential effects on inhibiting ACE in different tissues and suggest that the prolonged tissue ACE inhibition after a single oral dose of lisinopril may reflect targets involved in the hypotensive action of ACE inhibitors.     

Further studies on the effects of lesions in the rostral hypothalamus on gonadotropin secretion in the female rhesus monkey (Macaca mulatta).

Bilateral radiofrequency lesions were stereotaxically placed in the rostral hypothalamus of four adult female rhesus monkeys. These lesions resulted in extensive destruction of the ventromedial preoptic-anterior hypothalamic area (POA-AHA) and included the suprachiasmatic nucleus as well as, with the exception of one animal, the organum vasculosum of the lamina terminalis. In three of these four animals, gonadotropin surges similar to those observed before surgery were elicited in response to either a spontaneous increment in serum estrogen concentration or an estradiol benzoate injection. This stimulatory action of estradiol on LH and FSH release was not demonstratable in the remaining lesioned animal, but estradiol benzoate injections also failed to elicit a gonadotropin discharge in one of a series of five normal control animals. These findings fail to support the view that destruction of the ventromedial POA-AHA in this species compromises the ability of the hypothalamicohypophysial apparatus to respond to the positive feedback action of estradiol. The diurnal variation in serum cortisol concentration was not interrupted by placement of the lesions in the ventromedial POA-AHA.     

Evidence for the existence of atrial natriuretic factor-containing neurons in the rat brain

Immunoreactive atrial natriuretic factor- (ANF-)positive nerve fibers and cell bodies were observed in the preoptic area, hypothalamus, mesencephalon, and pons of rats. In colchicine-treated animals a large number of immunoreactive ANF-positive cell bodies were seen in the organum vasculosum of the lamina terminalis, in several hypothalamic nuclei (e.g. periventricular, arcuate, and ventral premammillary nuclei), and in the dorsolateral tegmental nuclei of the pons. Varicose nerve fibers containing ANF were generally observed in the vicinity of the cells. These findings indicate that a widespread network of ANF-containing neurons is present in the brain.     

Estrogen increases angiotensin II-induced c-Fos expression in the vasopressinergic neurons of the paraventricular nucleus in the female rat

Previous studies in female rats have shown that estrogen treatment attenuates angiotensin II (AngII)-induced water intake. The mechanism underlying this attenuation may be decreased responsiveness to AngII, as revealed by a reduction in AngII binding to the angiotensin type 1 (AT1) receptor in the subfornical organ (SFO). It has not been determined whether these changes in receptor binding translate into changes in neuronal activity that, in turn, may influence behavior. Therefore, an estrogen-modulated change in neuronal pathways relevant to AngII-induced water intake was tested in ovariectomized (OVX) female rats using immunohistochemistry for the immediate early gene c-Fos as a marker for neuronal activation. Third cerebroventricular injection of AngII (6 ng) induced intense c-Fos immunoreactivity in forebrain regions associated with fluid intake, including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the SFO, the supraoptic nucleus and the paraventricular nucleus (PVN). Forty-eight-hour estradiol (10 microg) administration to OVX female rats increased AngII-induced c-Fos labeling in the lateral magnocellular neurons of the PVN by 30% as compared to vehicle-treated controls. Double labeling neurons in the PVN with c-Fos and either vasopressin or oxytocin antisera revealed that estrogen increased AngII-induced c-Fos expression by 28%, specifically in vasopressinergic neurons. Such changes in neuronal activation may explain the estrogen modulation of AngII-induced water intake that has been previously reported; it may be due to increased water retention to maintain plasma osmolality or to induction of a pressor response.     

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 microg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10(-7) M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10(-7) M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood-brain barrier may have regulatory influences on fluid balance during pregnancy in rats.     

Estrogen receptor-immunoreactive glia, endothelia, and ependyma in guinea pig preoptic area and median eminence: electron microscopy.

The presence of estrogen receptors (ERs) in nonneural cells in brain, including glia, ependyma, and endothelia, has not previously been documented with electron microscopy. This study employed immunocytochemistry to investigate whether ER immunoreactivity (ER-ir) is present in glial, ependymal, or endothelial cells in the medial preoptic area (POA) and median eminence (ME) in the brain of gonadally intact female guinea pigs. Tissue sections through these regions were immunostained with monoclonal antibody H222 for ER localization using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogen. ER-ir cells were identified ultrastructurally by the presence of distinct spicule-like TMB crystals in nuclei. While neurons constituted the clear majority of ER-immunopositive cells, labeled astrocytes, ependyma, and endothelia were also present. Distinct intranuclear TMB crystals were present in astrocytes at the anterior pole of the POA within the preventricular periventricular nucleus, anterior compact subnucleus of the medial preoptic nucleus (MPNa), and organum vasculosum of the lamina terminalis, indicating ER-ir. In the MPNa, cell counts performed at the ultrastructural level revealed that 9.6% (15 of 156) of the astrocytes were ER-ir. To further explore the relationship of ERs with astrocytes, ER/glial fibrillary acidic protein (GFAP) double labeling experiments were performed using TMB and diaminobenzidine tetrahydrochloride for ER and GFAP localization, respectively. These studies verified the presence of ERs in astrocytes at the anterior pole of the POA and demonstrated the presence of ERs in GFAP-ir cells in the ME. Cell counts at the ME showed that 23 of 50 (46%) GFAP-ir cells were ER-ir. ER-ir was also present in scattered ependymal cells lining the third ventricle at the POA and overlying the ME. Typically, approximately four to eight ER-ir ependymal cells were present around the perimeter of the third ventricle, although occasionally small aggregations of greater numbers of labeled cells were observed. Both common ependyma and cells morphologically identified as tanycytes were ER-ir. Some endothelial cells and vascular smooth muscle cells also contained ERs. While approximately 11% of the vessels were lined by ER-ir cells in sections through the MPNa and preventricular periventricular nucleus, approximately 15% of the vessels were labeled in the organum vasculosum of the lamina terminalis. In the ME a greater percentage (59%) of the vessels contained ER-ir endothelial cells. Collectively, these results indicate that in addition to regulating the activity of neurons, estrogen may affect brain function through effects exerted on astrocytes, ependymal cells, and endothelial cells.