Angiotensin acts at the subfornical organ to increase plasma oxytocin concentrations in the rat

We have examined the effects of systemic angiotensin II (AII) on plasma oxytocin (OXY) concentrations in freely moving male Sprague-Dawley rats. We have also examined the role of the subfornical organ (SFO) as a CNS site at which circulating AII acts to influence secretion of this neurohypophysial peptide. OXY concentrations were measured by radioimmunoassay in plasma samples obtained by drawing blood samples through indwelling atrial catheters. In SFO intact animals (n = 8) AII infusion (1.0 microgram/kg/min) resulted in increases in plasma OXY concentrations from baseline values of 6.8 +/- 2.5 pg/ml to postinfusion concentrations of 44.9 +/- 11.9 pg/ml. In a second series of experiments electrolytic lesions were placed in the region of the SFO prior to testing the effects of AII infusion on OXY concentrations. Two further experimental groups were thus established according to the histologically verified location of lesions in either the rostral or caudal SFO. In the caudal SFO lesioned group AII infusion resulted in increases in plasma OXY concentrations from control values of 6.9 +/- 1.4 pg/ml to postinfusion levels of 45.1 +/- 9.8 pg/ml. These changes were not significantly different from the SFO intact group. In contrast rostral SFO lesions resulted in significantly elevated basal concentrations of OXY (17.4 +/- 3.4 pg/ml, n = 6) while postinfusion concentrations were found to be 22.8 +/- 4.9 pg/ml indicating that AII infusion was without effect following such lesions. These data are in accordance with the hypothesis that circulating AII acts at the SFO to influence SFO efferents which in turn activate OXY secreting neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. These neuroendocrine cells then release this peptide into the systemic circulation from the posterior pituitary.     

Angiotensin II mediated acth release in rat pituitary cell culture

Anterior pituitaries from normal rats were enzymatically dispersed and placed into monolayer cell culture in order to determine if and how angiotensin II (Ang II) mediates the $\text{in vitro}$ release of ACTH and other pituitary hormones. Ang II stimulated ACTH secretion in a time dependent fashion. This release occurred at physiologic concentrations of Ang II and was linearly correlated with the log dose of Ang II. One hour pretreatment of the cells with cycloheximide, a inhibitor of protein synthesis, significantly decreased the cellular ACTH secretory response to Ang II. Ang 11 did not mediate the release of LH nor of ADH, a proposed stimulator of ACTH secretion.     

Topographical distribution of acetylcholinesterase in the subfornical organ of the rat

Acetylcholinesterase (AChE) activity in the rat subfornical organ (SFO) was determined by the Koelle and Friedenwald method. Coronal sections showed a homogeneous reaction in the rostral region, which adopted a ring-like form in the anteromedial zone and a horseshoe pattern in the posteromedial zone. In the latter, AChE-specific positive neurons had been observed clearly in our sections. In sagittal sections the enzymatic reaction was intense in the anterior subependymal zone and dorsal region, and it continued caudally to the hippocampal area. The enzymatic activity was absent in the central region of the anteromedial zone and the subependymal region of the posteromedial zone. In sagittal sections the reaction was strongly positive in the periphery of the organ and negative in the posteromedial zone, near the choroid plexuses. Thus, we have shown that each region of the SFO presents a particular distribution of the AChE activity, related in some cases to neurons and in others to nerve fibers.     

Luliberin and somatostatin fiber-terminals in the subfornical organ of the rat

Summary With the aid of light- and electron- microscopic immunocytochemistry, somatostatin- and luliberin (LRF)-positive fibers can be demonstrated in the rat subfornical organ (SFO). Each of the neurohormones has a specific location: LRF in the lateral parts of the organ, and somatostatin in the center of the posterior zone. Common to both neurohormone-containing fibers is the pattern in which they reach the organ as well as the fact that their terminals are located in the perivascular spaces of fenestrated vessels, i.e., within the limited neurohemal regions of the organ. Since injection of India ink of different colors demonstrates that the capillary bed of the SFO is connected with the central capillaries of the choroid plexus, the question arises as to whether the neurohormones released in the area of the SFO influence the choroid plexus.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Activation of subfornical organ efferents stimulates oxytocin secretion in the rat

The effects of activation of subfornical organ (SFO) efferents on plasma oxytocin concentrations were examined in conscious freely moving male Sprague-Dawley rats. Blood samples were obtained through chronically implanted atrial catheters and SFO efferents were activated electrically using chronically implanted bipolar stimulating electrodes. Hormone concentrations were measured by radioimmunoassay, and experimental animals were assigned to one of 3 experimental groups according to histologically verified anatomical locations of stimulating electrodes in either the SFO, the hippocampal commissure (HC), or the medial septum (MS). Electrical stimulation in the SFO resulted in increased plasma concentrations of oxytocin from control values of 2.54 +/- 0.9 pg/ml, to a post-stimulation level of 65.6 +/- 27.0 pg/ml. In contrast, stimulation in immediately adjacent structures including HC and MS was found to be without effect on plasma concentrations of oxytocin. These studies provide the first definitive evidence that SFO efferents may play a significant role in controlling the secretion of oxytocin from the posterior pituitary.     

Effects of subfornical organ stimulation on respiration in the anesthetized rat

The studies reported here have demonstrated that electrical stimulation in the subfornical organ of the urethane-anesthetized rat elicits augmented breaths in over 90% of animals tested. Similar stimulation in immediately adjacent anatomical regions such as the hippocampal commissure was without effect. Detailed analysis of respiratory timing revealed no other significant respiratory effects of such subfornical stimulation.     

白藜芦醇抑制大鼠穹隆下器神经元放电

应用细胞外记录单位放电技术,在大鼠穹隆下器脑片上观察了白藜芦醇(resveratrol)对穹隆下器神经元放电的影响。实验结果如下:(1)给予白藜芦醇(1、5、10μmol/L)2min后,大多数穹隆下器神经元(60/65,92.3%)的自发性放电频率呈剂量依赖性降低;(2)预先用0.3mmol/L的L-glutamate灌流穹隆下器脑片,全部放电单位(12/12,100%)放电频率明显增加,表现为癫痫样放电,在此基础上灌流白藜芦醇(5μmol/L)2min,大多数脑片(10/12,83.3%)的癫痫样放电被抑制;(3)预先用L型钙通道开放剂BayK8644灌流,全部(8/8,100%)放电增加,在此基础上灌流白藜芦醇(5μmol/L)2min,其放电全部被抑制;(4)灌流一氧化氮合酶抑制剂NG-nitro-L-argininemethylester(L-NAME)50μmol/L,多数脑片(11/14,78.6%)放电明显增加,在此基础上灌流白藜芦醇(5μmol/L)2min,大部分神经元(9/11,81.8%)放电被抑制;(5)灌流大电导钙激活性钾通道阻断剂tetraethylammoniumchloride(TEA)1mmol/L后,大多数神经元(10/12,83.3%)放电增加,在此基础上灌流白藜芦醇(5μmol/L)2min,(9/10,90%)放电频率明显减低。以上结果提示:白藜芦醇能抑制大鼠穹隆下器神经元自发放电以及由L-glutamate、L-NAME、BayK8644和TEA诱发的放电,可能与白藜芦醇抑制L型钙通道以及促进一氧化氮的释放有关;似乎与大电导钙激活性钾通道无关。     

Postmortem persistence of acetylcholinesterase activity in the subfornical organ of the rat

Acetylcholinesterase (AChE) activity in the rat subfornical organ at 8, 12, 18, 24, 26, 28 and 30 h postmortem was evaluated by the Koelle and Friedenwald method. AChE activity was unchanged in animals 12 h postmortem. When the postmortem period was extended, enzymatic activity diminished, disappearing almost completely after 30 h.     

The stimulation of GABAB receptors increases serotonin release in the rat suprachiasmatic area

The action of γ-aminobutyric acid (GABA) and related compounds on the spontaneous release of newly synthesized [³H]5-hydroxytryptamine ([³H]5-HT) was studied in the suprachiasmatic area (SCA) using a superfusion system. GABA (10 μM) increased [³H]5-HT release from SCA by up to 190%. Bicuculline or picrotoxin (10 μM) failed to inhibit the stimulatory effect of GABA. Muscimol (10 μM), a GABA_A agonist, was ineffective, however β-p-chlorophenyl GABA, R(−)baclofen, enhanced over 200% the release of the indoleamine; this latter effect was stereospecific. RS baclofen was twice less potent than the R(−)isomer in increasing the [³H]5-HT release. S(+)baclofen failed to affect the release of the indoleamine, whereas it attenuated the effect of its enantiomer. The effect of R(−)baclofen was Ca²⁺ dependent and was abolished by tetrodotoxin (TTX).Taken together these results suggest that in the SCA, [³H]5-HT release is facilitated by the stimulation of GABA_B receptors. The possible localization of these receptors is discussed in the light of morphological data recently reported by Bosler et al. (1985) and results obtained after TTX application.     

Muscarinic modulation of GABAergic transmission to neurons in the rat subfornical organ

Cholinergic actions on subfornical organ (SFO) neurons in rat slice preparations were studied by using whole cell voltage- and current-clamp recordings. In the voltage-clamp recordings, carbachol and muscarine decreased the frequency of GABAergic inhibitory postsynaptic currents (IPSCs) in a dose-dependent manner, with no effect on the amplitudes or the time constants of miniature IPSCs. Meanwhile, carbachol did not influence the amplitude of the outward currents induced by GABA. Furthermore, carbachol and muscarine also elicited inward currents in a TTX-containing solution. From the current-voltage relationship, the reversal potential was estimated to be -7.1 mV. These carbachol-induced responses were antagonized by atropine. In the current-clamp recordings, carbachol depolarized the membrane with increased frequency of action potentials. These observations suggest that acetylcholine suppresses GABA release through muscarinic receptors located on the presynaptic terminals. Acetylcholine also directly affects the postsynaptic membrane through muscarinic receptors, by opening nonselective cation channels. A combination of these presynaptic and postsynaptic actions may enhance activation of SFO neurons by acetylcholine.     

Regulation of angiotensin II binding sites in the subfornical organ and other rat brain nuclei after water deprivation

1. Binding sites for angiotensin II have been localized in forebrain and brain-stem areas of water-deprived and control Sprague-Dawley rats, employing autoradiography with computerized microdensitometry. 2. Angiotensin II receptor sites were identified in the organum vasculosum of the lamina terminalis, subfornical organ, paraventricular nucleus, median preoptic nucleus, area postrema, nucleus of the solitary tract, and inferior olive. 3. After dehydration a significant increases in the concentration of angiotensin II receptors was detected only in the subfornical organ. Although there was an increased concentration of angiotensin II binding sites in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, and the paraventricular nucleus after dehydration, these changes did not reach statistical significance. Other brain nuclei investigated did not show differences in angiotensin II binding sites in the dehydrated rats compared to controls. 4. These results indicate that angiotensin II receptors in the subfornical organ may play an important role in fluid homeostasis during dehydration.     

Angiotensin II mediates beta endorphin like immunoactivity release in rat pituitary cell culture

The effect of angiotensin II (Ang II) on pituitary beta endorphin like immunoactivity (beta END-LI) release was studied in monolayer culture of normal rat pituicites. Ang II stimulated beta END-LI release into the culture media. This release of beta END-LI increased with longer incubation time and with higher doses of Ang II. The beta END-LI response was similar to the pattern of Ang II mediated ACTH release. Ang II stimulated beta END-LI release was blocked by cycloheximide and decreased by corticosterone (5 nmol/l). Successively higher concentrations of [SAR GLY]Ang II, a known Ang II antagonist, induced greater inhibition of Ang II stimulated beta END-LI release. Gel chromatography of pooled media from control and Ang II stimulated cells revealed three peaks of beta END-LI which migrated with the void volume, beta lipotropin (beta LPH) and beta endorphin. The relative amount of beta END-LI in these peaks [(BEND-LI peak + total beta END-LI in column) x 100%] from media of control and stimulated cells were as follows: (1) Void 7% and 19% (2) beta LPH 50% and 52% (3) beta endorphin 43% and 29%.     

Mechanisms underlying pressor response of subfornical organ to angiotensin II.

In urethane-anesthetized rats, microinjection of angiotensin II (AII) into either the subfornical organ (SFO), nucleus paraventricularis (NPV), or rostral ventrolateral medulla (RVL), respectively, all induced pressor responses, but the heart rate remained unchanged. Preinjection of [Sar1, Thr8]-angiotensin II (ST-AII, an AII antagonist) into bilateral NPV blocked the SFO-pressor response to AII. Bilateral RVL pretreated with ST-All markedly attenuated the pressor response of the SFO or NPV to AII. Hexamethonium or methyl atropine (IV) also reduced the SFO-pressor response. The results show that All can activate the SFO, NPV, and RVL successively, thereby inducing the pressor response; both excitation of sympathetic nerves and inhibition of the cardiac vagus are involved in this response.     

Morphology and physiology of capillary systems in subregions of the subfornical organ and area postrema

From recent morphological and physiological studies of capillaries, I shall review four new or revised concepts about blood-tissue communication in the subfornical organ (SFO) and area postrema (AP). First, the capillary systems of SFO and AP exhibit subregional differentiation correlated topographically with cytoarchitecture, densities of immunoreactivity for several peptides and amines, cellular sensitivity to neuroactive substances, afferent neural terminations, and tissue metabolic activity. Thus, contrary to frequent citations, the angioarchitecture and microcirculatory physiology of these small sensory nuclei are not homogeneous. Second, electron microscopic, morphometric, and topographical studies reveal that SFO contains three different types of capillary and AP has two. The differentiated capillary morphology appears to be well organized for specialized functions particularly in SFO subregions. No other body organ or small tissue region is known to have such capillary diversity, further highlighting the complex functions served by SFO. Third, pools of interstitial fluid (Virchow-Robin spaces) surrounding type I and III capillaries in SFO and AP may participate in the receptive properties of these organs as low-resistance pathways for rapid dispersion of blood-borne hormones inside their organ boundaries. The parenchymal walls of Virchow-Robin spaces appear to harbour metabolic mechanisms for hormones such as angiotensin II, and thus could vastly extend the effective blood-brain surface area of permeable capillaries in SFO and AP. Fourth, SFO and AP bear similar physiological characteristics of high blood volume, yet relatively low rates of blood flow. Accordingly, intracapillary blood velocity must be quite slow in these organs, and the duration of transit by blood and circulating messengers rather protracted. This feature of slow blood transit time likely compounds the sensory capability of SFO and AP, rendering increased contact time for blood-borne hormones to penetrate the permeable capillaries of these structures and interact with their known dense populations of receptors for several homeostatic substances involved in regulation of blood pressure and body fluids.     

Morphological plasticity of cultured astrocytes derived from the subfornical organ of the adult rat

The morphological plasticity of astrocytes from the subfornical organ of the adult rat has been examined using an explant culture preparation. Astrocytes migrate out of the explant and form a monolayer of amorphous, non-stellate cells. This non-stellate form was maintained when cultures were incubated in a HEPES buffered salt solution (HBSS) for 50 minutes. The fraction of cells that was stellate in these cultures was significantly increased when cultures were incubated in HBSS supplemented with forskolin (5 μM; but not 1,9-dideoxyforskolin) or with nitroprusside (10–100μM)indicating that elevation of intracellular cAMP or cGMP mediates stellation. The stellation responses induced by forskolin and by nitroprusside were blocked by inclusion of serum (0.5%) or of LY83,583 (10μM), an inhibitor of soluble guanylate cyclase, in the incubation medium. The relevance of the data to neuroglial plasticity in the subfornical organ in vivo is discussed.     

Angiotensin II blockade reduces radiation-induced proliferation in experimental radiation nephropathy

Total-body irradiation or renal irradiation is followed by a well-defined sequence of changes in renal function leading eventually to renal failure. Previous studies in a rat model have shown that inhibition of angiotensin-converting enzyme or blockade of angiotensin II receptors can prevent the structural and functional changes that occur after renal irradiation, and that these interventions are particularly important between 3 and 10 weeks after irradiation. We have now shown that in the same rat model, total-body irradiation induces proliferation of renal tubular cells (i.e., an increase in the number of cells staining positive for proliferating cell nuclear antigen) within 5 weeks after irradiation. Treatment with an angiotensin II receptor blocker delays this radiation-induced tubular proliferation and decreases its magnitude. Renal radiation also induces proliferation of glomerular cells, but the relative increase in glomerular proliferation is not as great as that seen in renal tubular cells, and the increase is not delayed or decreased by treatment with an angiotensin II receptor blocker. We hypothesize that angiotensin II receptor blockers exert their beneficial effect in radiation nephropathy by delaying the proliferation (and hence the eventual mitotic death) of renal tubular cells that have been genetically crippled by radiation.