Angiotensin III as well as angiotensin II regulates water flow through aquaporins in a clam worm

Angiotensin III has been reported to exist in various animals and tissues. The physiological role, however, is still unclear except that brain angiotensin III is a central regulator of vasopressin release. In this study, angiotensin III as well as angiotensin II enhanced an increase in body weight of clam worms of Perinereis sp. under a hypo-osmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo- and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms.     

Orexins and orexin receptors: implication in feeding behavior

Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.     

Angiotensin IV is a potent agonist for constitutive active human AT1 receptors. Distinct roles of the N-and C-terminal residues of angiotensin II during AT1 receptor activation

The octapeptide hormone, angiotensin II (Ang II), exerts its major physiological effects by activating AT(1) receptors. In vivo Ang II is degraded to bioactive peptides, including Ang III (angiotensin-(2-8)) and Ang IV (angiotensin-(3-8)). These peptides stimulate inositol phosphate generation in human AT(1) receptor expressing CHO-K1 cells, but the potency of Ang IV is very low. Substitution of Asn(111) with glycine, which is known to cause constitutive receptor activation by disrupting its interaction with the seventh transmembrane helix (TM VII), selectively increased the potency of Ang IV (900-fold) and angiotensin-(4-8), and leads to partial agonism of angiotensin-(5-8). Consistent with the need for the interaction between Arg(2) of Ang II and Ang III with Asp(281), substitution of this residue with alanine (D281A) decreased the peptide's potency without affecting that of Ang IV. All effects of the D281A mutation were superseded by the N111G mutation. The increased affinity of Ang IV to the N111G mutant was also demonstrated by binding studies. A model is proposed in which the Arg(2)-Asp(281) interaction causes a conformational change in TM VII of the receptor, which, similar to the N111G mutation, eliminates the constraining intramolecular interaction between Asn(111) and TM VII. The receptor adopts a more relaxed conformation, allowing the binding of the C-terminal five residues of Ang II that switches this "preactivated" receptor into the fully active conformation.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.     

The involvement of angiotensins in the control of prostatic epithelial cell proliferation in the rat.

The effects of captopril (the inhibitor of the angiotensin-converting enzyme) and of angiotensins II and IV (3-8 fragment of angiotensin II) on cell proliferation of the prostatic epithelium was investigated in the rat. The incorporation of bromodeoxyuridine into cell nuclei was used as an index of cell proliferation. It was found that the treatment with captopril resulted in the suppression of prostatic epithelial cell proliferation. The antiproliferative effect of captopril was reversed (at least partially) by a simultaneous treatment with either angiotensin II or angiotensin IV. The effects of angiotensins were not blocked by the administration of losartan--AT1 angiotensin receptor blocker. These findings suggest the involvement of angiotensins in the control of prostatic growth, acting via the receptors different from the AT1-subtype (presumably via AT4 receptors).     

A simplified radioimmunoassay for physiologically active angiotensin peptides [(1-8) octa- and (2-8) heptapeptides]

The availability of a sensitive and highly specific rabbit antiserum and the development of a peptide-extraction method employing glass beads permitted the evolution of a rapid reliable radioimmunoassay that measures the sum of the concentration of angiotensin II and its active metabolite, angiotensin III. At a dilution of 1:32,000 the antiserum is capable of measuring 1 fmol (1 pg) of angiotensin II. Cross reactivities of this antiserum, taking angiotensin II as 1.0, are: angiotensin III, 0.75; angiotensin-(3-8) hexapeptide, 0.11; angiotensin I, 0.006; angiotensin-(1-14) tetradecapeptide, 0.0001. The recovery of angiotensin II added to hormone-free plasma was 73 +/- 2% [mean +/- standard deviation (SD), n = 20]. When 0.9 ml of plasma was extracted, the minimal concentration of angiotensin II and III that could be quantified was 4 fmol/ml. When larger volumes of plasma were extracted, sensitivity was enhanced. Plasma blanks were zero. Intra-assay variability was 7.6% SD and interassay variability was 11.7% SD. Angiotensin II and III concentration in venous plasma of normal volunteers on an ad libitum diet was 15 +/- 8 fmol/ml (mean +/- SD, range less than 4 to 35 fmol/ml). The plasma of a patient with primary aldosteronism had an unmeasurable value (less than 4 fmol/ml). Posture, converting enzyme inhibition, and renal artery stenosis resulted in expected changes of angiotensin concentration.     

Enzymatic formation of angiotensins II and III in the hindlimb circulation of dogs

Experiments were performed in 14 anesthetized dogs to (1) to determine if the reductions in hindlimb blood flow produced by [des-Asp1] angiotensin I were due to its local enzymatic (kininase II) conversion to angiotensin III and (2) to quantitate the extent of conversion of angiotensin I to angiotensin II and of [des-Asp1] angiotensin I to angiotensin III in the hindlimb circulation. Graded doses of these peptides were administered as bolus injections directly into the left external iliac artery while measuring flow in this artery electromagnetically. Dose-response relationships were determined before and during the inhibition of kininase II activity with captopril or antagonism of angiotensin receptor sites with [Ile7] angiotensin III. Captopril inhibited the vasoconstrictor responses to angiotensin I and [des-Asp1] angiotensin I, but did not affect the responses to angiotensins II or III, or norepinephrine. [Ile7] angiotensin III inhibited the vasoconstrictor responses to all four angiotensin peptides but did not alter the responses to norepinephrine. These findings indicate that the hindlimb vasoconstrictor responses to [des-Asp1] angiotensin I were due to the local formation of angiotensin III. The extent of conversion of [des-Asp1] angiotensin I to angiotensin III that occurred in one transit through the hindlimb arterial circulation was estimated to be 36.7%, which was not different from the estimated 36.4% conversion of angiotensin I to angiotensin II. We conclude that angiotensin I and [des-Asp1] angiotensin I are converted to their respective vasoactive forms (angiotensins II and III) to a similar extent in the hindlimb circulation via the action of kininase II.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

Formation of angiotensin III by angiotensin-converting enzyme.

Angiotensin III is formed from des-Asp1 -angiotensin I by angiotensin-converting enzyme. The Km (11 muM) of the reaction is one-third of that for the conversion of angiotensin I into angiotensin II. As suggested by the Km values, bradykinin, peptide BPP9a and angiotensins II and III are better inhibitors of the formation of angiotensin II than of the formation of angiotensin III.     

Influence of angiotensins (I, II, & III), bradykinin and arachidonic acid on renomedullary PGE production in vitro.

Renomedullary tissue from rabbit or rat was incubated with angiotensin I, II, III, arachidonic acid, bradykinin, indomethacin and meclofenamate to study their effect on PGE2 production. Arachidonic acid and bradykinin enhanced PGE2 production significantly. Indomethacin and meclofenamate inhibited PGE2 production by more than 70%. Angiotensin I, II and III did not influence PGE2 production. These results suggest that bradykinin and arachidonic acid stimulate PGE2 production by a direct cellular action whereas the angiotensins do not.     

Monophasic and biphasic effects of angiotensin II and III on norepinephrine uptake and release in rat adrenal medulla.

Angiotensin II and III have hypertensive effects. They induce vascular smooth muscle constriction, increase sodium reabsorption by renal tubules, stimulate the anteroventral third ventricle area, increase vasopressin and aldosterone secretions, and modify catecholamine metabolism. In this work, angiotensin II and III effects on norepinephrine uptake and release in rat adrenal medulla were investigated. Both angiotensins decreased total and neuronal norepinephrine uptake. Angiotensin II showed a biphasic effect only on evoked neuronal norepinephrine release (an earlier decrease followed by a later increase), while increasing the spontaneous norepinephrine release only after 12 min. On the other hand, angiotensin III showed a biphasic effect on evoked and spontaneous neuronal norepinephrine release. Both angiotensins altered norepinephrine distribution into intracellular stores, concentrating the amine into the granular pool and decreasing the cytosolic store. The results suggest a physiological biphasic effect of angiotensin II as well as angiotensin III that may be involved in the modulation of sympathetic activity in the rat adrenal medulla.     

Angiotensin II induces prostaglandin E(2) release in human gingival fibroblasts

We investigated the effect of angiotensin II on prostaglandin E(2) release in human gingival fibroblasts. Stimulation of human gingival fibroblasts with angiotensin II elicited prostaglandin E(2) release in a concentration- and time-dependent manner. Angiotensin III also induced prostaglandin E(2) release, but the effect was weaker than that of angiotensin II. Angiotensin II- and angiotensin III-induced prostaglandin E(2) release was inhibited by AT(1) receptor antagonist FR-130,739, but not AT(2) receptor antagonist PD-123,319. Angiotensin II evoked an increase in intracellular Ca(2+) in fura-2-loaded human gingival fibroblasts. These results suggest that angiotensin II functions as a physiological mediator via Ca(2+)-mobilizing AT(1) receptor activation in human gingival fibroblasts.     

Immunohistochemical localization of the angiotensin-(1–7) receptor Mas in the murine forebrain

Apart from the well-known biologically active angiotensin II, other biologically active angiotensins have been discovered, including angiotensin IV and angiotensin-(1–7). Some years ago, we and others discovered that the Mas proto-oncogene encodes a receptor that is essential for angiotensin-(1–7) signaling. Angiotensin-(1–7) is not only expressed in the periphery but also within the brain. Based on that, we examined the distribution of Mas within the murine brain, using an antibody directed against the 3^rd cytoplasmic loop of the receptor protein. Strongest Mas protein expression was detected in the dentate gyrus of the hippocampus and within the piriform cortex. However, Mas protein expression is not restricted to these areas, since Mas immunopositive neurons were also seen in different parts of the cortex, hippocampus, amygdala, basal ganglia, thalamus and hypothalamus. Based on the expression of Mas protein in the cortex and the limbic system, angiotensin-(1–7) signaling may play a role in synaptic plasticity, learning, memory and emotion, as has been described for angiotensin II and IV.     

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones.

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.     

Angiotensin II and III upregulate body fluid volume of the clam worm Perinereis sp. via angiotensin II receptors in different manners

Angiotensin III (Ang III) as well as angiotensin II (Ang II) suppressed body weight loss of the clam worm Perinereis sp. under a hyper-osmotic condition, and enhanced body weight gain under a hypo-osmotic condition. Under a drying condition where the water inflow from outside the body was eliminated, Ang II suppressed body weight loss, but Ang III did not. Under these conditions, angiotensins I, IV, and (1–7) had no effect, and saralasin blocked the effects of Ang II and Ang III. It is concluded that Ang II and Ang III upregulate body fluid volume of the clam worm via Ang II receptors in different ways.     

The brain renin-angiotensin system: location and physiological roles

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.     

Biphasic actions of angiotensin IV on renal blood flow in the rat

Our aim was to investigate the changes in renal blood flow during brief exposure of the renal vasculature to angiotensin IV (Ang IV). Total renal blood flow was measured by electromagnetic flowmetry in pentobarbital-anesthetized male Sprague Dawley rats. Intrarenal injection of Ang I, Ang II and Ang III produced a dose-dependent vasoconstriction. In contrast, Ang IV and Ang-(3-10) produced a dose-dependent rapid vasoconstriction (lasting seconds) followed by a transient vasodilatation (lasting minutes). The biphasic response to Ang IV was unchanged in rats pre-treated with captopril, whereas the Ang-(3-10) response was abolished implying that its vasoactive activity was due to the generation of Ang IV. The vasodilatory component of Ang IV was unaffected by indomethacin. The biphasic vasoactive response of Ang IV was unaffected by divalinal-Ang IV (AT(4) receptor antagonist) or PD 123319 (AT(2) receptor antagonist), but greatly reduced by losartan or L-158,809 (AT(1) receptor antagonists). These results suggest that Ang IV is distinct from other angiotensins in that it possesses non-prostaglandin mediated renal vasodilatory activity that is apparently linked to the renal vascular AT(1) receptor.     

The CNS renin-angiotensin system

The renin-angiotensin system (RAS) is one of the best-studied enzyme-neuropeptide systems in the brain and can serve as a model for the action of peptides on neuronal function in general. It is now well established that the brain has its own intrinsic RAS with all its components present in the central nervous system. The RAS generates a family of bioactive angiotensin peptides with variable biological and neurobiological activities. These include angiotensin-(1–8) [Ang II], angiotensin-(3–8) [Ang IV], and angiotensin-(1–7) [Ang-(1–7)]. These neuroactive forms of angiotensin act through specific receptors. Only Ang II acts through two different high-specific receptors, termed AT1 and AT2. Neuronal AT1 receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and the secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT1 receptors. Among the many potential effects mediated by stimulation of AT2 are neuronal regeneration after injury and the inhibition of pathological growth. Ang-(1–7) mediates its antihypertensive effects by stimulating the synthesis and release of vasodilator prostaglandins and nitric oxide and by potentiating the hypotensive effects of bradykinin. New data concerning the roles of Ang IV and Ang-(1–7) in cognition also support the existence of complex site-specific interactions between multiple angiotensins and multiple receptors in the mediation of important central functions of the RAS. Thus, the RAS of the brain is involved not only in the regulation of blood pressure, but also in the modulation of multiple additional functions in the brain, including processes of sensory information, learning, and memory, and the regulation of emotional responses.