Defective Renal Angiotensin III and AT2 Receptor Signaling in Prehypertensive Spontaneously Hypertensive Rats.

Background Previous studies demonstrated that angiotensin (Ang) III , not Ang II , is the predominant endogenous agonist for Ang type-2 receptor ( AT 2R)-induced natriuresis in normal rats, and that hypertensive 12-week-old spontaneously hypertensive rats ( SHR ) lack natriuretic responses to Ang III . This study tested whether prehypertensive SHR already have defective Ang III -induced natriuresis and determined possible mechanisms. Methods and Results Female and male normotensive 4-week-old SHR and Wistar Kyoto rats were studied after 24-hour systemic AT 1R blockade. Left kidneys received 30 minute renal interstitial infusions of vehicle followed by Ang III (3.5, 7.0, 14, and 28 nmol/kg per min; each dose for 30 minutes). Right kidneys received vehicle infusions. In 4-week-old Wistar Kyoto rats, renal interstitial Ang III increased urine sodium (Na+) excretion but failed to induce natriuresis in 4-week-old SHR . Renal Ang III levels were similar between Wistar Kyoto rats and SHR , making increased Ang III degradation as a possible cause for defective natriuresis in SHR unlikely. In Wistar Kyoto rats, renal interstitial Ang III induced translocation of AT 2Rs to apical plasma membranes of renal proximal tubule cells. Simultaneously, Ang III induced retraction of the major Na+ transporter Na+-H+ exchanger-3 ( NHE -3) from apical membranes and internalization of Na+/K+ ATP ase ( NKA ) from basolateral membranes of renal proximal tubule cells. Consistent with NHE -3 and NKA retraction, Ang III increased pS er552- NHE -3 and decreased pS er23- NKA . In contrast, in SHR , intrarenal Ang III failed to induce AT 2R translocation, NHE -3 or NKA retraction, pS er552- NHE -3 phosphorylation, or pS er23- NKA dephosphorylation. Conclusions These results indicate impaired Ang III / AT 2R signaling as a possible primary defect in prehypertensive SHR .

Inhibitory effect of angiotensin (1-7) on angiotensin III-induced nociceptive behaviour in mice.

We have previously demonstrated that the intrathecal (i.t.) administration of angiotensin (Ang) II into mice produces a nociceptive behaviour consisting of scratching, biting and licking accompanied by the phosphorylation of p38 MAPK in the spinal cord, which was mediated through AT1 receptors. Both the p38 MAPK phosphorylation and subsequent nociceptive behaviour were attenuated by the i.t. co-administration of Ang (1-7), an N-terminal fragment of Ang II, that acted via Mas receptors. On the other hand, a C-terminal fragment of Ang II, namely Ang III, was also shown to induce a nociceptive behaviour by acting upon AT1 receptors on spinal astrocytes and neurons, and was found to be more potent than Ang II. However, the inhibitory effect of Ang (1-7) on the Ang III-induced nociceptive behaviour remains unclear. Thus, here we examined whether Ang (1-7) can attenuate the Ang III-induced nociceptive behaviour and activation of spinal p38 MAPK. The i.t. administration of Ang (1-7) (1-100fmol) dose-dependently attenuated the Ang III (1pmol)-induced nociceptive behaviour in mice. Moreover, the inhibitory effect of Ang (1-7) at a dose of 100fmol was prevented by A779 (30fmol), a Mas receptor antagonist. Western blot analysis showed that the phosphorylation of p38 MAPK induced by the i.t. administration of Ang III (1pmol) was also attenuated by Ang (1-7) (100fmol), and this inhibition was prevented by A779 (30fmol). Furthermore, we showed that in the lumbar superficial dorsal horn, Mas receptors are expressed in neurons and microglia but absent from astrocytes. Together, these results suggest that the i.t. administration of Ang (1-7) attenuates the nociceptive behaviour and accompanying p38 MAPK phosphorylation induced by Ang III, and that this effect is likely mediated through Mas receptors on spinal neurons.

p38 Mitogen-activated protein kinase is stimulated by both angiotensin II and angiotensin III in cultured rat astrocytes.

CONTEXT: Previously we showed that angiotensin (Ang) II and Ang III induced phosphorylation of ERK1/2 and JNK mitogen-activated protein (MAP) kinases in rat astrocytes. OBJECTIVES: To determine whether these peptides induce p38 MAP kinase in astrocytes. MATERIALS AND METHODS: We used brainstem astrocytes as a model system to determine whether Ang II and Ang III induce p38 MAP kinase protein phosphorylation. RESULTS: Treatment of astrocytes with increasing concentrations of both peptides caused a dose-dependent increase in p38 MAP kinase phosphorylation. The effect of Ang II and Ang III was maximal at 10 nM and 100 nM concentrations, respectively. The effects of the peptides were rapid occurring within minutes of treatment. There was a significant difference in the ability of the peptides to induce p38 MAP kinase phosphorylation. The ability of Ang II to induce p38 MAP kinase was almost twice than that of Ang III, suggesting that Ang II was more potent than Ang III in this effect. Ang AT1 receptor mediated the actions of the peptides since pretreatment with losartan prevented p38 MAP kinase phosphorylation by Ang II and Ang III. In addition, blockade of Ang II metabolism to Ang III with the aminopeptidase A inhibitor glutamate phosphonate was ineffective in ameliorating Ang II phosphorylation of p38 MAP kinase, suggesting that Ang II directly stimulated p38 MAP kinase phosphorylation. CONCLUSION: These findings provide insight into the molecular nature of the actions of these peptides and offer a possible mechanism by which these Ang peptides physiological and possibly pathological actions occur in astrocytes.

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor.

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH2O to 7.5 cmH2O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 µM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT2) receptor antagonist but not by AT1 or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81±10.19% but Ang II decreased plasma ANP level by 30.41±7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT2 receptor/PI3K/Akt/nitric oxide/PKG pathway.

Central ventilatory and cardiovascular actions of angiotensin peptides in trout.

In the brains of teleosts, angiotensin II (ANG II), one of the main effector peptides of the renin-angiotensin system, is implicated in various physiological functions notably body fluid and electrolyte homeostasis and cardiovascular regulation, but nothing is known regarding the potential action of ANG II and other angiotensin derivatives on ventilation. Consequently, the goal of the present study was to determine possible ventilatory and cardiovascular effects of intracerebroventricular injection of picomole doses (5-100 pmol) of trout [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, ANG IV, and ANG 1-7 into the third ventricle of unanesthetized trout. The central actions of these peptides were also compared with their ventilatory and cardiovascular actions when injected peripherally. Finally, we examined the presence of [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, and ANG IV in the brain and plasma using radioimmunoassay coupled with high-performance liquid chromatography. After intracerebroventricular injection, [Asn(1)]-ANG II and [Asp(1)]-ANG II two ANG IIs, elevated the total ventilation through a selective stimulatory action on the ventilation amplitude. However, the hyperventilatory effect of [Asn(1)]-ANG II was threefold higher than the effect of [Asp(1)]-ANG II at the 50-pmol dose. ANG III, ANG IV, and ANG 1-7 were without effect. In addition, ANG IIs and ANG III increased dorsal aortic blood pressure (P(DA)) and heart rate (HR). After intra-arterial injections, none of the ANG II peptides affected the ventilation but [Asn(1)]-ANG II, [Asp(1)]-ANG II, and ANG III elevated P(DA) (50 pmol: +80%, +58% and +48%, respectively) without significant decrease in HR. In brain tissue, comparable amounts of [Asn(1)]-ANG II and [Asp(1)]-ANG II were detected (ca. 40 fmol/mg brain tissue), but ANG III was not detected, and the amount of ANG IV was about eightfold lower than the content of the ANG IIs. In plasma, ANG IIs were also the major angiotensins (ca. 110 fmol/ml plasma), while significant but lower amounts of ANG III and ANG IV were present in plasma. In conclusion, our study suggests that the two ANG II isoforms produced within the brain may act as a neurotransmitter and/or neuromodulator to regulate the cardioventilatory functions in trout. In the periphery, two ANG IIs and their COOH-terminal peptides may act as a circulating hormone preferentially involved in cardiovascular regulations.

Influence of dietary sodium on the blood pressure and renal sympathetic nerve activity responses to intracerebroventricular angiotensin II and angiotensin III in anaesthetized rats.

The regulation of blood pressure and sympathetic outflow by the brain renin-angiotensin system in animals subjected to raised or lowered dietary Na(+) intake is unclear. This study compared the mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) responses to intracerebroventricular (i.c.v.) infusion of angiotensin II (AngII) and III (AngIII) before and after peripheral V(1) receptor blockade (V(1)B) in alpha-chloralose-urethane-anaesthetized rats fed a low (0.03%, LNa(+)), normal (0.3%, NNa(+)) or high Na(+) diet (3.0%, HNa(+)) from 4 to 11 weeks of age. The rise in MAP 2 min post AngII i.c.v. was greater in HNa(+) (14 +/- 3 mmHg) versus LNa(+) (8 +/- 1 mmHg, P < 0.05) and after AngIII i.c.v. in HNa(+) (14 +/- 3 mmHg) versus NNa(+) (6 +/- 1 mmHg, P < 0.05) and LNa(+) (7 +/- 1 mmHg, P < 0.05). The MAP responses to AngII and AngIII i.c.v. were abolished after V(1)B in LNa(+), but were only attenuated in HNa(+). In NNa(+), V(1)B blunted the MAP responses to AngII and abolished those to AngIII. The MAP remained elevated 30 min after AngII in all groups, but returned to baseline levels 15 min after AngIII in NNa(+) and HNa(+) (P < 0.01). Twenty minutes after i.c.v. AngII, RSNA rose above baseline in HNa(+) (112 +/- 1%), a response not observed in the LNa(+) and NNa(+) groups. Twenty minutes post AngIII i.c.v., RSNA was elevated in both HNa (109 +/- 2%) and NNa(+) (109 +/- 2%). After V(1)B, RSNA rose only in the HNa(+) group 15 min post AngIII infusion (109 +/- 1%). Together, these findings: (1) suggest that HNa(+) intake augments the MAP and RSNA responses to i.c.v. AngII and AngIII; (2) highlight an important role for peripheral V(1) receptors during these responses; and (3) differentiate the effects of AngII and AngIII on blood pressure and RSNA.

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice.

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.

[Effector peptides of the brain renin-angiotensin system in central mechanisms of learned and natural forms of drinking behavior in rats].

Central mechanisms of angiotensin involvement in initiation and realization of operant forms of drinking behavior were investigated. It was suggested that intracerebroventricular microinjection of angiotensin-II and angiotensin-IIl specifically affected the learned forms of drinking behavior. The experiments demonstrated that [des-Asp1]-angiotensin-I produced only the natural forms of drinking behavior. Angiotensins modulated specific forms of thirst-associated behavior such as exploring, grooming, and ingestive behavior. Injections of AT1 receptor antagonist losartan were associated with acute water intake decrease and sharp operant behavior inactivation.

Roles of brain angiotensins II and III in thirst and sodium appetite.

The current study examined the effects of intracerebroventricular (icv) infused aminopeptidase-resistant analogs of angiotensin II (AngII) and angiotensin III (AngIII) on thirst and sodium appetite. The analogs, [D-Asp1D-Arg2]AngII and [D-Arg1]AngIII, were further protected from degradation by pretreatment with the aminopeptidase A inhibitor, EC33, or the aminopeptidase N inhibitor, PC18. Prior to icv infusions, rats were sodium depleted with furosemide, followed by the angiotensin-converting enzyme inhibitor captopril, to block endogenous angiotensin formation. Both angiotensin analogs, at either of the two doses, were capable of eliciting fluid intakes of water and 0.3 M NaCl. Water and saline intakes were increased to a similar extent by 125 and 1250 pmol of [D-Asp1D-Arg2]AngII. [D-Arg1]AngIII produced a dose-dependent increase in water intake, whereas saline intake was equivalently increased by the 125 and 1250 pmol infusions. Pretreatment with EC33 or PC18 decreased water and saline intakes in response to [D-Asp1D-Arg2]AngII, while pretreatment with PC18 altered the time course of the [D-Arg1]AngIII-induced water and saline intakes. The ability of both inhibitors to decrease, but not completely block, AngII analog-induced intakes, coupled with the altered time course of the responses induced by the AngIII analog in the presence of PC18, supports the hypothesis that both AngII and AngIII are active ligands in brain angiotensin-mediated thirst and sodium appetite. However, these results do not resolve the primary question of whether conversion of AngII to AngIII is a prerequisite to dipsogenic and salt appetite responses in the brain.

Angiotensin II enhances thrombosis development in renovascular hypertensive rats.

There is an increased number of in vitro evidence that angiotensin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT(1) receptor and Ang II metabolites: angiotensin III (Ang III) and angiotensin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT(1) antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT(1) receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites - Ang IV.

ANG III induces expression of inducible transcription factors of AP-1 and Krox families in rat brain.

In addition to rapid responses comprising increases in blood pressure, drinking, and stimulation of natriuresis, ANG II induces the expression of transcription factors (TF) in the central nervous system. The ANG II metabolite ANG III (ANG 2-8) has been demonstrated to exert physiological effects similar to those of ANG II. We aimed to determine 1) whether ANG III induces TF expression in the brain, 2) which ANG II (AT) receptor subtype is involved, and 3) whether the two peptides, ANG II and ANG III, differ in their efficacy to stimulate TF expression. ANG II (100 pmol), ANG III (100 pmol), or vehicle was injected into the lateral brain ventricle of conscious rats alone or in combination with the AT(1) receptor antagonist losartan (10 nmol), the AT(2) receptor antagonist PD-123319 (5 nmol), or the aminopeptidase inhibitor amastatin (10 nmol). Similar to ANG II, ANG III induced the expression of c-Fos, c-Jun, and Krox-24 in four brain regions, subfornical organ, median preoptic area, paraventricular nucleus, and supraoptic nucleus of the hypothalamus, with the same efficacy. This effect was AT(1) receptor mediated. Pretreatment with amastatin reduced the expression of TF in response to ANG II, indicating that this expression is partly mediated by ANG III. Interestingly, the AT(2) receptor antagonist PD-123319 alone slightly enhanced the expression of c-Fos, c-Jun, and Krox-24 in different populations of neurons of the paraventricular nucleus. These data indicate that different populations of neurons in the paraventricular nucleus are tonically inhibited by AT(2) receptors under physiological conditions.

Effects of angiotensin II and AIII microinjections into the zona incerta after intra- and extracellular fluid loss.

Our recent results showed that angiotensin II or III (AII, AIII) microinjected into the zona incerta (ZI) significantly increased water intake. The most effective doses of AII and AIII were also defined. The two neuropeptides had their effects differently on drinking via different receptors. AII bound to AT(1) that was blocked by AT(1) receptor antagonist Losartan and the effect of AIII was eliminated by prior application of AT(2) receptor antagonist PD 123319. After different hydrational challenges, the effects of AII and AIII in the ZI have never been experimented, however. In the present experiments, the previously defined effective doses of AII (100 ng) or AIII (200 ng) were microinjected into the ZI after different types of challenges: (1). lowered thirst motivation when animals ingested approximately 40% of their daily fluid need during the consequent 60-min-daily-drinking period before the injection, (2). 48-h water deprivation, (3). intracellular dehydration and (4). extracellular dehydration. In all of the cases, incertally injected AII increased the animals' water ingestion. While Losartan could block these effects, PD 123319 was ineffective. Experiments were repeated by AIII, but in none of the cases differences were experienced between the groups. The finding that following hydrational challenges water intake increased only after AII injections and it could be blocked only by Losartan suggests that AII and AT(1) receptor play a pivotal role in the ZI in maintaining the body water balance.

Effects of truncated angiotensins in humans after double blockade of the renin system.

Angiotensins different from ANG II exhibit biological activities, possibly mediated via receptors other than ANG II receptors. We studied the effects of 3-h infusions of ANG III, ANG-(1-7), and ANG IV in doses equimolar to physiological amounts of ANG II (3 pmol. kg-1. min-1), in six men on low-sodium diet (30 mmol/day). The subjects were acutely pretreated with canrenoate and captopril to inhibit aldosterone actions and ANG II synthesis, respectively. ANG II infusion increased plasma angiotensin immunoreactivity to 53 +/- 6 pg/ml (+490%), plasma aldosterone to 342 +/- 38 pg/ml (+109%), and blood pressure by 27%. Glomerular filtration rate decreased by 16%. Concomitantly, clearance of endogenous lithium fell by 66%, and fractional proximal reabsorption of sodium increased from 77 to 92%; absolute proximal reabsorption rate of sodium remained constant. ANG II decreased sodium excretion by 70%, potassium excretion by 50%, and urine flow by 80%, whereas urine osmolality increased. ANG III also increased plasma aldosterone markedly (+45%), however, without measurable changes in angiotensin immunoreactivity, glomerular filtration rate, or renal excretion rates. During vehicle infusion, plasma renin activity decreased markedly ( approximately 700 to approximately 200 mIU/l); only ANG II enhanced this decrease. ANG-(1-7) and ANG IV did not change any of the measured variables persistently. It is concluded that 1) ANG III and ANG IV are cleared much faster from plasma than ANG II, 2) ANG II causes hypofiltration, urinary concentration, and sodium and potassium retention at constant plasma concentrations of vasopressin and atrial natriuretic peptide, and 3) a very small increase in the concentration of ANG III, undetectable by usual techniques, may increase aldosterone secretion substantially.

Stimulation of collagen gel contraction by angiotensin II and III in cardiac fibroblasts.

OBJECTIVE: The aim of the present study was to investigate whether angiotensin II (Ang II), angiotensin III (Ang III) or Ang II (2-8), angiotensin IV (Ang IV) or Ang II (3-8) and Ang II (1-7), Ang II (4-8), Ang II (5-8) and Ang II (1-4) can stimulate collagen gel contraction in cardiac fibroblasts in serum-free conditions. METHODS: Cardiac fibroblasts (from male adult Wistar rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel in a Dulbecco's Modified Eagle's Medium, with or without foetal bovine serum, for one, two or three days. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Collagen gel contraction was characterised by a decrease in the gel area. RESULTS: Ang II dose-dependently stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. Telmisartan completely blocked the Ang II-induced collagen contraction by cardiac fibroblasts. PD 123319 and des-Asp(1)-Ile(8)-Ang II had no effect on the Ang II-induced collagen contraction by cardiac fibroblasts. Ang III also stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. des-Asp(1)-Ile(8)-Ang II and telmisartan completely blocked the Ang III-induced collagen gel contraction by cardiac fibroblasts. des-Asp(1)-Ile(8)-Ang II, however, had no effect on the Ang II-induced collagen gel contraction by cardiac fibroblasts. Ang IV and Ang II (4-8), (5-8), (1-7) and (1-4), however, had no effect on collagen gel contraction by cardiac fibroblasts. Addition of telmisartan, PD 123319 or des-Asp(1)-Ile(8)-Ang II alone did not affect collagen gel contraction by cardiac fibroblasts. CONCLUSION: Our data demonstrate that the effects of Ang II on the collagen gel contraction by adult rat cardiac fibroblasts in serum-free conditions are Ang II type 1(AT(1))-receptor- mediated, because they are abolished by the specific AT(1)-receptor antagonist, telmisartan, and not by the AT(2)-receptor antagonist PD 123319 or by the Ang III antagonist des-Asp(1)-Ile(8)-angiotensin. The Ang III- stimulated contraction of collagen by cardiac fibroblasts is completely blocked by the Ang III receptor antagonist, des-Asp(1)-Ile(8)-angiotensin II, and by telmisartan.

Angiotensin III: a central regulator of vasopressin release and blood pressure.

Among the main bioactive peptides of the brain renin-angiotensin system, angiotensin (Ang) II and AngIII exhibit the same affinity for type 1 and type 2 AngII receptors. Both peptides, injected intracerebroventricularly, cause similar increases in vasopressin release and blood pressure. Because AngII is converted in vivo to AngIII, the identity of the true effector is unknown. This review summarizes new insights into the predominant role of brain AngIII in the control of vasopressin release and blood pressure and underlines the fact that brain aminopeptidase A, the enzyme forming central AngIII, could constitute a putative central therapeutic target for the treatment of hypertension.

Pressor and renal effects of intracerebroventricularly administered angiotensins II and III in rats.

AIMS: Experiments were performed to assess the effects of intracerebroventricular (ICV) angiotensin (ANG) III on blood pressure and renal function in rats with normal and high sodium intake and to compare these effects with those produced by ICV ANG II. METHODS: Male Sprague-Dawley rats on a normal sodium (0.3%) diet and a normal sodium diet plus 1% NaCl as drinking water were administered ANG II and ANG III ICV through a chronically implanted cannula. Blood pressure and renal clearance function responses were measured before and during peptide administrations. The effect of ICV ANG III on the renal efferent nerve activity was also evaluated. RESULTS: ICV injections of ANG II and ANG III at 5 pmol in rats on a normal sodium diet did not significantly alter the blood pressure, but significantly increased renal plasma flow, glomerular filtration rate, urine flow, and absolute and fractional excretions of sodium and potassium. Increased doses of ANG II and III (10, 50 and 100 pmol) significantly increased blood pressure and further enhanced these renal functional indices. Central ANG-III-induced increases in blood pressure and renal functional indices were not significantly different from those produced by ANG II at each corresponding dose. The pressor and renal effects of ANG III were blunted by a specific antagonist, Ile(7)-ANG III. ICV administration of ANG III decreased the renal efferent nerve activity. In rats with dietary NaCl loading, ICV injections of ANG II and III also significantly enhanced renal function. CONCLUSIONS: Centrally administered ANG III is as potent as ANG II in causing pressor and renal effects in rats on normal and high sodium intake. As ANG II, brain ANG III reduced renal efferent nerve activity which may be partly accounted for the augmented renal function.

Inhibition of vasopressinergic neurons by central injection of a specific aminopeptidase A inhibitor.

The brain angiotensin (Ang) system plays an important role in the central control of vasopressin release. Using EC33, a selective aminopeptidase A inhibitor which blocks the metabolism of Ang II in Ang III, we previously reported that vasopressin release was under the control of Ang III and not Ang II. To determine accurately the action of EC33, the effects of intracerebroventricular injection of Ang peptides or EC33 on extracellular unit activity of vasopressinergic neurons in the supraoptic nucleus of urethane-anaesthetized rats were examined. Angiotensin II (15-30 ng) or Ang III (15 ng) increased the firing rate of all neurons tested. Conversely, EC33 (10 microg) reduced or completely abolished (30-60 microg) the basal firing rate for 4-6 min in all eight neurons tested. EC33 (30 microg) also inhibited the activity induced by 30 ng Ang II. It was concluded that the observed activity of Ang II required its conversion to Ang III and that endogenous Ang III may exert a tonic control on the basal firing level of vasopressinergic neurons.

Functional characterization of caudal hypoglossal neurons by spectral patterns of neuronal discharges in the rat.

This study evaluated the spectral characteristics of neuronal discharges in the caudal hypoglossal nucleus and their physiological relevance in adult, male Sprague Dawley rats which were anaesthetized and maintained with pentobarbital sodium. Based on auto-spectral analysis of extracellular single-neuron activity, three spectral patterns were identified in the spontaneous discharges of hypoglossal neurons. Neurons that exhibited a rhythmic pattern manifested a concentrated peak in the auto-spectrogram that corresponded to the mean discharge rate. A majority of hypoglossal neurons displayed the modulated pattern, which was manifested either as scattered power densities (wide-band modulated pattern) or with a peak frequency component that was different from the mean discharge rate (narrow-band modulated pattern). Neurons that exhibited a mixed pattern displayed both rhythmic and modulated spectral patterns. Cross-spectral analysis further revealed that respiratory modulation constituted a major physiological influence on caudal hypoglossal neurons. The respiratory modulated pattern, however, could be converted to a mixed pattern in the presence of a central dipsogen, angiotensin III. The results suggest that the spectral patterns of neuronal discharges in caudal hypoglossal neurons represent manifestations of multiple physiological information, including that regarding respiration and dipsogenesis, which is encoded in these neurons. It was also shown that this information may only be revealed by auto-spectral and cross-spectral analysis of neuronal discharge signals.

Circulating Na+/K+-ATPase inhibitors: effects of neuropeptides, volume expansion and salt loading in conscious rats.

1. In mammalian plasma, many different inhibitors of Na+/K(+)-ATPase are present, but it is not clear whether their net effect on NA+/K(+)-ATPase activity changes during the regulation of electrolyte and fluid balance. We studied Na+/K(+)-ATPase inhibition by plasma extracts in conscious rats during short- and long-term body fluid regulation. 2. Male, adult, conscious, freely moving Wistar rats were subjected to one of the following protocols: (i) intracerebro-ventricular (i.c.v.) injections of angiotension II (AngII; 1, 10 and 100 ng), the AngII receptor antagonist losartan (1 microgram), atrial natriuretic peptide (ANP-III; 1 microgram) or isotonic saline (IS); (ii) intra-arterial (i.a.) injections of IS (6 or 10 mL), hypertonic saline (HS; 1.2% NaCl, 5 mL) or hypertonic plasma expander (HPS; 3.5% hetastarch in HS, 5 mL); or (iii) a low salt-high salt-low salt diet sequence (0.18/1.8/0.18% NaCl chow for 5 days each with controls receiving 0.18% NaCl on all days). Bodyweight, the intake of food and water, urine volume and Na+ concentration and weight of faeces were determined daily. Plasma samples were withdrawn repeatedly throughout the respective protocols, extracted on C18-reversed phase columns and assayed for their effect on the activity of different Na+/K(+)-ATPase preparations. 3. The inhibition of rat brain Na+/K(+)-ATPase by plasma extracts was not significantly changed by i.c.v. injection of AngII, losartan, ANP-III and IS within the observation period (30 min from respective stimuli). Similarly, no significant changes occurred after acute volume expansion by i.a. injection of IS or HS within 120 min; upon HPS, however, Na+/K(+)-ATPase inhibition was decreased by approximately 20% (P < 0.05), probably due to passive dilution. During the high-salt diet, fluid retention was effectively counteracted by an adaptive increase of urinary sodium excretion. Throughout the protocol, inhibition of pig brain Na+/K(+)-ATPase by plasma extracts did not differ significantly between groups. 4. It is concluded from these results that the short- or long-term control of body fluids in conscious rats is not associated with systematic changes in Na+/K(+)-ATPase inhibition by plasma factors.

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.