Central Angiotensinergic Pathway Research Articles

Abstract P142: Regulation of Brain Derived Neurotrophic Fractor (BDNF) Expression by Angiotensin II in the Adrenals and the Brain

Circulating Ang II activates central angiotensinergic pathways, and an amplifying aldosterone (aldo)-MR-ENaC-AT 1 R neuromodulatory pathway which are critical for several forms of hypertension. We hypothesized that in addition Ang II increases BDNF expression in both the CNS and adrenal which plays a balancing role against Ang II excitatory actions. In the 1 st exp, Wistar rats were sc infused with Ang II at the high dose of 500 ng/kg/min for 7days. In the 2 nd exp, rats were treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II at the low dose of 150 ng/kg/min, or sc Ang II with high salt diet for 14 days. In the 3 rd exp, MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT 1 R blocker (losartan) or vehicles (Veh) were icv infused combined with Ang II-salt. In the 4 th exp, rats were sc infused with aldo (1μg/hr) with saline as drinking fluid. mRNA levels of BDNF were measured by real-time qPCR. In the adrenals, Ang II dose-related increased BDNF mRNA, and Ang II-salt further enhanced BDNF mRNA. High dose Ang II increased BDNF mRNA in the RVLM but not in the PVN. Central blockades markedly decreased Ang II-salt induced BDNF expression in the adrenal cortex. In contrast, saline alone or aldo-saline decreased BDNF mRNA in the adrenal cortex with no effect in the PVN. Central blockades lower Ang II-salt induced BDNF expression in the adrenal, indicating that a central regulatory mechanism plays a role in adrenal BDNF expression. Activation of BDNF by Ang II -modulated by salt-, may provide an important balancing mechanism both centrally and peripherally for Ang II associated hypertension.

Chronic treatment with mianserin prevents DOCA–salt hypertension in rats—evidence for the involvement of central 5-HT2 receptors

Central 5-HT2A receptors have been implicated in central volume control by activating a central angiotensinergic pathway to cause the release of vasopressin. Interestingly, to induce DOCA–salt hypertension in rats vasopressin release is required. Thus the present experiments were carried out to determine whether continuous blockade of these receptors over 20 days, with the non-selective 5-HT2 receptor antagonist mianserin would prevent the development of deoxycorticosterone acetate (DOCA)–salt hypertension. Mianserin, given i.c.v. 90 or 60 μg twice daily for 20 days prevented the development of hypertension in conscious rats receiving DOCA–salt but did not affect blood pressure in rats on salt alone. Further, the dose of 30 μg given i.c.v. twice daily had no effect nor did the vehicle, polyethylene glycol (PEG), on the development of the hypertension. Mianserin 90 μg twice daily i.c.v. was also shown to prevent the increase in fluid intake, urinary flow and sodium excretion caused by DOCA–salt treatment. These data indicate that this action of mianserin is not due to an intrinsic hypotensive action but an action which involves interference with the mechanism by which DOCA–salt treatment causes hypertension. Thus the data overall support the view that to induce hypertension with DOCA–salt a central 5-HT-containing pathway needs to be activated, which then activates 5-HT2 receptors to cause the release of vasopressin which has previously been shown to be responsible for the initiation of DOCA–salt treatment hypertension.

Effect of dietary sodium intake on central angiotensinergic pathways

The role of central angiotensinergic pathways in the cardiovascular regulation has been examined using the microinjection of angiotensin peptides and angiotensin receptor antagonists. However, in such studies, neither the overall nor the local level of activity of the renin–angiotensin system is generally known. Herein, physiological changes in the endogenous level of activity of the renin–angiotensin system were produced by alterations in the dietary sodium intake. Microinjection of the angiotensin II AT 1 receptor antagonists losartan or candesartan into the rostral ventrolateral medulla produced the bradycardic, depressor and renal sympathoinhibitory responses which were greater in low sodium diet rats with stimulated activity of the renin–angiotensin system than in high sodium diet rats with suppressed activity of the renin–angiotensin system activity. The renal sympathoexcitatory responses to activation of the paraventricular nucleus by microinjection of bicuculline, known to be dependent on the excitatory synaptic inputs to the rostral ventrolateral medulla mediated by AT 1 receptors, were greater in low sodium diet rats than in high sodium rats. These observations support the view that physiologically regulated angiotensin peptides of the brain origin exert a local paracrine or autocrine action on sites that influence the renal sympathetic nerve activity.

Central cardiovascular regulation and 5-hydroxytryptamine receptors

Evidence is provided to support the view that central 5-HT 1A and 5-HT 2 receptors are the major receptor subtypes important in cardiovascular regulation. Data are also provided to implicate 5-HT 1B/1D/1F receptors in central cardiovascular regulation. Activation of 5-HT 2 receptors generally causes sympathoexcitation and a rise in blood pressure and this is mainly mediated by 5-HT 2A receptors. However, presympathetic vasomotor neurones located in the hindbrain (RVLM), controlling sympathetic outflow to the heart, are not activated in the same way as other presympathetic vasomotor neurones, although activation of 5-HT 2 receptors located in the midbrain can activate sympathetic outflow to the heart. Furthermore, at least in the rat, these midbrain 5-HT 2A receptors are also responsible for the release of vasopressin by activation of a central angiotensinergic pathway. The ability of vasopressin directly and/or indirectly to modify renal sympathetic outflow involves the activation of central 5-HT 2B receptors, which in turn, when activated via the i.c.v. route, can cause selective renal sympathoexcitation. Evidence is also provided which indicates that the reflex control of parasympathetic outflow to the heart and to other organs involves central 5-HT 1A receptors located in the vicinity of these preganglionic vagal neurones. Finally, 5-HT 3 receptors are implicated in the afferent regulation of central sympathetic and parasympathetic tone.

Brain angiotensinergic pathways mediate renal nerve inhibition by central hypertonic NaCl in conscious sheep

The renal sympathetic responses to infusion of hypertonic solutions into the lateral cerebral ventricles were investigated in conscious sheep. Intracerebroventricular infusion of artificial cerebrospinal fluid (CSF) containing 0.6 M NaCl, at 1 ml/h for 20 min, reduced renal sympathetic nerve activity (RSNA) by 81 +/- 5% (n = 6, P < 0.001). Plasma renin concentration also fell (P < 0.05), whereas arterial pressure increased by 6.4 +/- 0.7 mmHg (P < 0.01). Intracerebroventricular hypertonic sorbitol (0.9 M in CSF at 1 ml/h) had no effect. The AT1 receptor antagonist losartan (1 mg/h) abolished the plasma renin and arterial pressure responses to intracerebroventricular hypertonic saline and significantly reduced the fall in RSNA to 17 +/- 10% (P < 0.001). During intracerebroventricular hypertonic saline, the baroreflex relation of RSNA to diastolic pressure was shifted to the left and that to central venous pressure was abolished compared with control relations obtained by manipulating pressure with intravenous phenylephrine. These findings indicate that 1) RSNA is inhibited by a central mechanism that senses high sodium (or perhaps chloride) concentration rather than hypertonicity; 2) this inhibition occurs independently of reflexes from high- and low-pressure baroreceptors, although these may enhance the inhibition; and 3) inhibition of RSNA by hypertonic saline involves a central angiotensinergic pathway.

2317 Disruption of progesterone-induced facilitation of female rat sexual behavior by ventral tegmental and ventral premammillary lesions

The role of central angiotensin II (AII) in the shaking stress-induced adrenocorticotropic hormone (ACTH), plasma catecholamine secretion and pressor response were investigated using conscious rats. We also studied whether or not vasopressin (VP) is involved in the shaking stress-induced pressor response. The shaking stress caused significant elevations in plasma ACTH, catecholamine, and systolic blood pressure. Intra-third ventricular administration of the AII antagonist, Sar1, Ile8-angiotensin II (saralasin) significantly attenuated pressor response and plasma noradrenaline elevation but not plasma ACTH elevation. Pretreatment with the vascular-type VP receptor (V1) antagonist, d(CH2)5Tyr(Me)AVP, did not attenuate pressor response nor plasma catecholamine elevation. These results indicate that the central angiotensinergic pathway at least partly mediates the shaking stress-induced activation of the sympathetic nervous system without VP involvement, and that central AII does not mediate the ACTH secretion evoked by shaking stress.

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN II MEDIATED BY AT1 AND AT2 RECEPTORS IN THE BRAIN

1. Autoradiographic binding studies have shown that the AT(1) receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT(1) receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord. 2. While there are differences between species, AT(2) receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat. 3. Circulating AngII acts on AT(1) receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking. 4. Centrally administered AngII may act on AT(1) receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion. 5. Recent evidence shows that centrally administered AT(1) antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses. 6. Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT(1) receptors and play a role in the function of sympathetic pathways maintaining arterial pressure.

NaCl injections in brain induce natriuresis and blood pressure responses sensitive to ANG II AT1 receptors.

In the present study we tested the hypothesis that the natriuretic and pressor effects of intracerebroventricularly (icv) injected hypertonic saline involve a central angiotensinergic pathway. All experiments were performed in conscious Wistar rats. Bolus injections of hypertonic saline (0.19, 0.23, 0.30, and 0.60 M icv; injection volume 5 microliters) induced a concentration-dependent increase of renal sodium excretion without affecting urinary flow. The increase in renal sodium excretion after the two highest saline concentrations was accompanied by significant increases in mean arterial blood pressure (MAP). Pretreatment with the angiotensin (ANG) AT1 receptor antagonist, losartan (5 micrograms icv), reduced the natriuretic effect of 0.23 and 0.30 M saline but did not affect the natriuresis induced by 0.60 M saline. The increase in MAP after 0.30 and 0.60 M saline icv was markedly attenuated by intracerebroventricular pretreatment with losartan. Our results demonstrate the involvement of a central angiotensinergic mechanism in the natriuretic and pressor responses to hypertonic saline. In addition to the ANG II-mediated natriuresis, an additional natriuretic mechanism, independent of ANG II and associated with the saline-induced pressor effect, seems to be recruited with increasing concentrations of saline in the cerebrospinal fluid.

Fos induced in brain of spontaneously hypertensive rats by angiotensin II and co-localization with AT-1 receptors

The induction of Fos-like immunoreactivity (FLI) by peripheral administration of angiotensin II (Ang II) was used to determine whether central activation was greater in spontaneously hypertensive rats (SHR) than in normotensive WKY and outbred Wistar controls. FLI was induced in the same brain regions (circumventricular organs and neurosecretory hypothalamic cell groups) in all three groups of rats, but the FLI in several of these regions was markedly less in WKY than in either SHR or Wistar. This reduced responsiveness in supraoptic and paraventricular nuclei was selective to Ang II, because the FLI induced in these nuclei by hypertonic NaCl did not differ between groups. We also report that a considerable number of cells in the SON and PVH expressing FLI to these stimuli show immunostaining with an antibody to the AT-1 Ang II receptor. These data indicate that central angiotensinergic pathways may be more sensitive in SHR than WKY, and that WKY are less sensitive than outbred Wistars.

Effects of central losartan on plasma renin and centrally mediated natriuresis

Because intracerebroventricular (ICV) infusion of hypertonic saline or angiotensin II (Ang II) both induce water drinking, vasopressin secretion, natriuresis and increased arterial pressure, the possibility that common neural pathways mediate responses to ICV Ang II and hypertonic saline has been investigated. This was done by testing the effect of ICV infusion of the Ang II antagonist losartan on the natriuretic and pressor responses to ICV hypertonic NaCl in sheep. The effect of ICV losartan on plasma renin concentration (PRC) was also investigated. Infusion of losartan (1 mg/hr) into a lateral ventricle prevented both natriuretic and pressor responses to infusion of 0.6 mol/liter NaCl into a lateral ventricle at 1 ml/hr. In another experiment, ICV losartan at 1 mg/hr caused a pronounced increase in the PRC of Na-depleted sheep, while ICV Ang II at 3 micrograms/hr decreased PRC. The results suggest that: (i) a central angiotensinergic pathway may mediate osmoregulatory responses to centrally administered hypertonic saline, and (ii) a central angiotensinergic pathway may have a tonic inhibitory influence on renin secretion in Na-depleted animals.

Central angiotensinergic system and hypertonic resuscitation from severe hemorrhage

Single injections of 4 ml/kg hypertonic NaCl (7.5%) resuscitate dogs from severe blood loss (40-45 ml/kg). Mechanisms involve osmolarity-dependent volume expansion, increased myocardial contractility, and vasodilation. The role of central angiotensinergic pathways in the hemorrhage-hypertonic resuscitation interaction was investigated through experiments performed on male pentobarbital sodium-anesthetized dogs bled to, and held at, 40 mmHg for 30 min. Dogs were treated with 4 ml/kg of 7.5% NaCl or 32 of 0.9% NaCl iv preceded by intracerebroventricular (ICV) injections of 150 micrograms saralasin, 20 micrograms arginine vasopressin inhibitor (AVPI), or 10 micrograms morphine. ICV saralasin and morphine inhibited the full recovery response to hypertonic NaCl, whereas AVPI had no such effect. Saralasin did not inhibit the recovery from hemorrhagic shock produced by large volume isotonic saline reexpansion. These data demonstrate an interaction between the central angiotensin system and small volume hypertonic resuscitation from severe hemorrhagic shock but not between this central system and large volume isotonic reexpansion of circulatory volume. In contrast, the central vasopressinergic system does not appear to be similarly involved.

Role of central angiotensinergic mechanism in shaking stress-induced ACTH and catecholamine secretion

The role of central angiotensin II (AII) in the shaking stress-induced adrenocorticotropic hormone (ACTH), plasma catecholamine secretion and pressor response were investigated using conscious rats. We also studied whether or not vasopressin (VP) is involved in the shaking stress-induced pressor response. The shaking stress caused significant elevations in plasma ACTH, catecholamine, and systolic blood pressure. Intra-third ventricular administration of the AII antagonist, Sar 1, Ile 8-angiotensin II (saralasin) significantly attenuated pressor response and plasma noradrenaline elevation but not plasma ACTH elevation. Pretreatment with the vascular-type VP receptor (V1) antagonist, d(CH 2) 5Tyr(Me)AVP, did not attenuate pressor response nor plasma catecholamine elevation. These results indicate that the central angiotensinergic pathway at least partly mediates the shaking stress-induced activation of the sympathetic nervous system without VP involvement, and that central AII does not mediate the ACTH secretion evoked by shaking stress.

Improved immunohistochemical staining of angiotensin II in rat brain using affinity purified antibodies

Recent immunohistochemical studies that have sought to detect angiotensin II/III (AII/AIII) immunoreactive material in the brain have been forced to rely on a small number of antisera because most AII/AIII antibodies have unexplainably proved unsuitable for immunohistochemistry. Although extremely useful tools, these antisera have suffered from high background staining. The purpose of this study was to re-examine and characterize the staining using the most popular AII/AIII antiserum (Denise) before and after purification on an AII CH-sepharose affinity column. The use of crude AII/AIII antiserum resulted in the staining of large varicosities and cell bodies. Fibres were all but invisible owing to extensive background staining. In contrast, the purified antibodies yielded little background staining and produced a discrete staining of AII/AIII fibres with small varicosities in the paraventricular-hypophysial pathway and of cell bodies of large hypothalamic neurones. In addition punctate staining demarcated the perikarya of some neurones and resembled boutons containing immunoreactive AII/AIII. Biochemical and histochemical analysis of the crude antiserum, the affinity purified antibodies and other fractions off the sepharose column demonstrated that a large portion of the total staining (various types of background) seen with crude antiserum and column fractions was not to AII/AIII or several angiotensin-derived fragments. Furthermore, successful preabsorption blanks for the purified antibodies could only be achieved with AII coupled through its N-terminal, suggesting that these purified antibodies reacted best with conjugated angiotensin in the fixed tissue. In total the results of this study indicate that the background staining seen with crude antiserum is not to AII/AIII. The use of affinity purified antibodies greatly enhances resolution, enabling one to visualise even small fibres in rats not treated with colchine, and should improve our ability to develop accurate maps of central angiotensinergic pathways.

Comparison of Angiotensin II Staining in Rat Brain using Affinity Purified and Crude Antisera

The use of affinity purified ANG II antiserum as opposed to crude antiserum greatly enhanced the staining resolution in rat brain. This improved resolution was due to a complete loss of background staining and an apparent increase in specific staining that was totally blockable by preabsorption. With the purified antibody it was easily possible to visualise the finest fibres in rats not treated with colchicine. Furthermore, the improved technique permitted a clearer visualisation of an ANG II-like immunoreactive product in cell bodies. This use of affinity purified antibody should greatly facilitate the mapping of central angiotensinergic pathways.