Radioimmunoassay for immunoreactive [des-Leu10]-angiotensin I

A specific radioimmunoassay for the angiotensin-derived peptide [des-Leu10]-angiotensin I (AI-dL) is described. Antisera obtained from rabbits injected with immunogen prepared by coupling bovine beta-thyroglobulin to the peptide with carbodiimide were specific to this peptide and did not recognise related angiotensin peptides such as AI, AII, AIII, nor did they recognise other peptides such as bradykinin, substance P, bombesin or dynorphin(1-8). Immunoreactive AI-dL was detected for the first time in the plasma of rats and humans following purification by HPLC at concentrations of 78 and 40 pg/ml, respectively. Concentrations of AI-dL are increased following chronic administration of captopril to rats.     

Angiotensin and thirst: studies with a converting enzyme inhibitor and a receptor antagonist

Although exogenous angiotensin is recognized as a potent dipsogen, the participation of endogenous angiotensin in thirst has not been well established. To investigate this question, we produced thirst in rats by relative cellular dehydration (hypertonic NaCl injection), or hypovolemia (hyperoncotic polyethylene glycol injection). An angiotensin receptor antagonists (sar(1)-ala(8)- angiotensin II, P-113), or a converting enzyme inhibitor (SQ, 20, 881, SQ) given to thirsty rats by intracerebroventricular (IVT) or peripheral routes. P-113 infused i.v. (10 μg/kg/min) or injected IVT (10 μg) did not alter the drinking response to either thirst stimulus. The latter treatment reduced the drinking response to 50 ng of IVT angiotensin II (p < 0.005). SQ given i.m. (2 mg/kg), IVT (2 × 50 μg), or both routes did not alter relative cellular dehydration thirst. Injection of SQ IVT did not alter hypovolemic thirst, whereas a significantly (p < 0.005) enhanced response occured after i.m. SQ. The enhanced response was not observed when animals were given both IVT and i.m. SQ. The IVT treatment with SQ markedly reduced (P < 0.005) drinking after 50 ng IVT angiotensin I. The data demonstrate that inhibition of angiotensin receptors or converting enzyme does not prevent appropriate drinking responses to primary thirst stimuli. Thus, if angiotensin participates in these endogenous thirst drives, its role is not an absolute requirement.     

Acute abdominal vagotomy reduces drinking to peripheral but not central angiotensin II

Rats were tested two or three days after bilateral abdominal vagotomy or a laparotomy control procedure for their drinking responses to subcutaneous (1 mg-kg-1) or intracerebroventricular (100 ng) injections of angiotensin II. Vagotomy delayed the initiation of drinking and decreased 60-min water intake after subcutaneous, but not after intracerebroventricular, angiotensin II. This is the shortest postoperative interval in which the decrease in drinking after systemic injection of angiotensin II by abdominal vagotomy has been observed. The failure of vagotomy to decrease the response to intracerebroventricular angiotensin II demonstrates that the deficit after subcutaneous injection was not a nonspecific effect of recent vagotomy. These results, therefore, suggest that the abdominal vagus is necessary for normal drinking in response to circulating angiotensin II. Furthermore, the selective and acute onset of the deficit is consistent with the loss of a specific, rather than tonic facilitatory, vagal mechanism for drinking after elevation of circulating angiotensin II levels. Finally, the results imply that the physiological mechanisms which mediate the drinking responses to central and peripheral angiotensin are not identical.     

Identification of the angiotensin II receptor in rat mesenteric artery.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.     

Antisense oligonucleotide inhibition of angiotensinogen in the brains of rats and sheep

Phosphorothioated antisense oligodeoxynucleotides (ODNs) that were complementary to various parts of the rat or sheep mRNA encoding angiotensinogen were synthesized by conventional techniques. Their effectiveness as blockers of angiotensinogen synthesis in the brain was tested by bioassay. This involved measuring the effect of centrally administered antisense ODNs on water drinking that occurred in response to intracerebroventricular injection of hog renin. Renin-induced drinking requires brain angiotensinogen for the generation of angiotensin I and then angiotensin II to stimulate thirst. Intracerebroventricular injection of an 18-mer antisense ODN (0.5 microg twice in 24 h) complementary to the 5'-end start codon for rat angiotensinogen mRNA caused a pronounced inhibition of renin-induced drinking. This effect appeared to be specific for this region of the codon because antisense ODNs directed against other regions of rat angiotensinogen mRNA were ineffective, and renin-induced drinking was not inhibited by intracerebroventricular injection of scrambled or mismatched sequences of the effective ODN or by intraperitoneal injection of it. Intracerebroventricular injection of antisense ODN (0.5 microg twice in 24 h) did not inhibit appetite or affect water drinking in response to some other dipsogenic stimuli, thus demonstrating the specificity of its action against renin-induced drinking. By contrast, intracerebroventricular administration of 625 microg of an antisense ODN directed against the corresponding 5'-end start codon region of sheep angiotensinogen mRNA did not inhibit intracerebroventricular renin-induced drinking in sheep. These data show that while intracerebroventricularly administered antisense may be used effectively in rodents, the method is not necessarily applicable in larger mammals.     

Hypothalamic alpha-adrenergic blockade modifies drinking and blood pressure responses to central angiotensin II in conscious rats

These experiments investigated in the awake rat the involvement of noradrenergic projections to the rostral hypothalamus in the drinking and pressor responses elicited by intracerebroventricular (i.c.v.) injections of 25 ng of angiotensin II. Phentolamine mesylate in doses of 2.5-125 micrograms injected into the rostral hypothalamus produced a dose-dependent depression of both the drinking and pressor responses elicited by i.c.v. administration of angiotensin II. A paradoxical increase in heart rate was associated with a decrease in pressor responses with increasing doses of phentolamine. This response was due to tissue injections, since pretreatment by injecting 12.5 micrograms of phentolamine into the ventricle did not block either the cardiovascular or drinking responses to i.c.v. injections of angiotensin II. Yohimbine (0.33-3.3 micrograms), DL-propranolol (25 micrograms), and atenolol (25 micrograms) did not, but prazosin (0.7 microgram) did significantly alter the pressor responses. Although yohimbine also was without effect on drinking, prazosin reduced the drinking responses. These results suggest that alpha 1-adrenergic receptors in the rostral hypothalamus are involved in the control of both the drinking and pressor responses elicited by i.c.v. injections of angiotensin II. In the case of propranolol and atenolol, beta-adrenergic receptors altered only the drinking response in a nonspecific manner by eliciting competing behaviors. Whether they are involved in modifying the drinking response only remains to be demonstrated.     

The dipsogenic potency of peripheral angiotensin II

The effective intravenous dose of angiotensin II for the induction of drinking in the rat has been reduced to a physiologically reasonable level of approximately 10 ng/min/rat or 25 ng/min/kg. It can be as low as 4 ng/min/rat when the natural state of thirst is more closely simulated with concurrent cellular dehydration. These doses are not different from those employed intravenously in the intact mammal to produce the hormone's pressor response. In addition: (i) the drinking produced by the hormone occurs sooner and more reliably and is greater in volume when the hormone is combined with mild cellular dehydration, and (ii) the drinking is blocked by a specific competitive inhibitor of angiotensin II, but (iii) is not affected by diurnal cycle. The evidence supports the suggestion that angiotensin II is a normal participant in drinking provoked by hypovolemia.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Biochemical and functional changes during immunization of rats with angiotensin II

Angiotensin-converting enzyme (ACE) activity in serum and some brain areas, level of angiotensin I in the blood and drinking behaviour during immunization of rats against conjugate of angiotensin II with bovine serum albumin (BSA) were studied. The results show that an increase in antibodies against angiotensin II was correlated with elevated ACE activity in serum. There was a distinct tendency towards elevated level of angiotensin I in the blood. After a 6 month's immunization ACE-activity was reduced twofold to threefold in midbrain and hypothalamus-thalamus. During immunization water-uptake was increased by 40-45%.     

Impaired angiotensin-induced drinking in spontaneously hypertensive rats

The drinking response during the 30 minutes following intracerebroventricular injection of angiotensin II (AII) (1 to 200 ng) was compared in male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto control rats (WK). SHR drank significantly less than WK at the 10, 50 and 100 ng doses. In contrast, responses to intracerebroventricular carbachol and intraperitoneal hypertonic saline were not different between SHR and WK. These agents are believed to induce drinking by mechanisms independent of angiotensin. Binding of I¹²⁵-labelled AII to particles prepared from the hypothalamus, thalamus, septum and midbrain (HTSM) region was measured in one month old male and two month old female SHR and their respective age matched WK controls. No differences were found in binding between SHR and WK of either sex. These data demonstrate an impairment of drinking responses in the SHR which seem to be specific for angiotensin. This finding supports the hypothesis that the CNS angiotensin system might be abnormal in these animals.     

Kainic acid lesions of the median preoptic nucleus: effects on angiotensin II induced drinking and pressor responses in the conscious rat

Input to the nucleus medianus of the preoptic region has been suggested to be involved in both the drinking and pressor responses elicited by the central administration of angiotensin II. Evidence in support of this suggestion has been gained principally from electrical lesion experiments. This lesion procedure does not differentiate between the cells of the region and fibers coursing through the region. To test the hypothesis that cells in this region are involved in both the pressor and drinking responses elicited by central administration of angiotensin II, injections of kainic acid were made to induce lesions of the cells, while sparing fibers of passage. Drinking and blood pressure responses were determined pre- and post-lesion in the chronically instrumented awake rat. Injections of 50 ng angiotensin II in a 2-microL volume into a lateral cerebral ventricle of the conscious rat elicited pronounced drinking and pressor responses with a latency of 3-5 min. Lesions of the median preoptic region produced by injecting 1.0 microgram of kainic acid in 0.25 microL for 15 s attenuated or blocked the drinking response and increased the latency to drink induced by central injections of angiotensin II. However, kainic acid lesions did not significantly alter the pressor responses produced by angiotensin II administration. These results suggest that cells in the median preoptic region are involved in the drinking response but do not participate in the pressor response elicited by angiotensin II administration into a lateral cerebral ventricle of the conscious rat.     

Effects of angiotensin I and II and their interactions with some prostanoids in perfused human umbilical arteries

In perfused human umbilical arteries both angiotensin I and II induced vasoconstriction with a monophasic response. Angiotensin I and II induced vasoconstrictions at doses greater than or equal to 10(-8) M and 10(-9) M respectively. Captopril inhibited the angiotensin I response while the angiotensin II receptor blocker Sar1-Ala8 AII inhibited the effect of both angiotensins. PGI2 attenuated the angiotensin II response in a dose dependent pattern. PGE2 and PGF2 alpha in concentrations below the critical levels for creating pressure responses per se, also attenuated the angiotensin II response. The cyclooxygenase inhibitor indomethacin potentiated the angiotensin II response indicating that endogenous production of prostanoids is of importance in the modulation of angiotensin effects.     

Big angiotensin-25: A novel glycosylated angiotensin-related peptide isolated from human urine

The renin–angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.     

Angiotensin, thirst, and sodium appetite: retrospect and prospect.

The fact that drinking in response to some hypovolemic stimuli was attenuated by nephrectomy but not by ureteric ligation led to the suggestion that the renal renin-angiotensin system may play a role in hypovolemic thirst. The isolation of a thirst factor from the kidney and the demonstration that this substance was renin supported the hypothesis. Subsequently, it was shown that the effects of renin on drinking were mediated through angiotensin II, which proved to be a potent dipsogenic substance when administered systemically or injected directly into the brain. Recently, it has been shown that angiotensin II, infused intravenously or through the carotid artery at rates that produce increases in plasma angiotensin II levels similar to those that occur in mild sodium depletion, causes the water-replete animal to drink. This discovery establishes that angiotensin is a physiological stimulus to drinking but it leaves open the question of the extent of the involvement of renal renin in normal thirst. Other unsolved problems are the role of cerebral isorenin in angiotensin thirst and its relationship with renal renin, and in view of its stimulating action on sodium intake when infused into the brain, whether angiotensin plays a significant role in sodium appetite.     

Effect of DuP 753, a nonpeptide angiotensin II receptor antagonist, on the drinking responses to acutely administered dipsogenic agents in rats.

The present studies examine the effect of the nonpeptide angiotensin II (AII) type 1 receptor antagonist, DuP 753, on water intake in rats treated with dipsogenic stimuli, which are thought to induce drinking via release of renin and subsequent formation of AII. Subcutaneous administration of DuP 753 in doses that are known to inhibit drinking induced by AII failed to inhibit the water intake of rats following subcutaneous administration of the beta-adrenoceptor agonist isoproterenol. The peptide antagonist1 Sar, 8Ileu-AII, which blocks both AII type 1 and AII type 2 receptors, also failed to inhibit isoproterenol-induced drinking, suggesting that neither subtype is involved in this drinking response. Additional studies verified previous reports that acute subcutaneous administration of both the beta-adrenoceptor antagonist propranolol and the angiotensin I-converting enzyme inhibitor captopril could block the drinking response to subcutaneous administration of isoproterenol. Subcutaneous administration of DuP 753 also failed to inhibit the drinking responses to subcutaneous administration of serotonin, 5-hydroxytryptophan, hypertonic saline, and polyethylene glycol. However, central intraventricular administration of DuP 753 inhibited the drinking response to subcutaneous administration of isoproterenol. The results are discussed in terms of the importance of AII in mediating isoproterenol-, serotonin-, and 5-hydroxytryptophan-induced water intake and suggest a need to readdress this mechanism.     

Central integration of cardiovascular and drinking responses elicited by central administration of angiotensin II: divergence of regulation by the ventral tegmental area and nucleus accumbens

Previous studies had implicated the involvement of the ventral tegmental area and its dopamine projections to the nucleus accumbens in goal-directed behavior. This study investigated whether or not the GABAergic inputs to the ventral tegmental area and, in turn, dopaminergic input to the nucleus accumbens from the ventral tegmental area modify drinking and cardiovascular responses elicited by central administration of angiotensin II. Injections of 25 ng of angiotensin II into a lateral cerebral ventricle of the rat elicited water intakes averaging 7-8 mL in 15 min with latencies usually less than 3 min. Pretreatment of the nucleus accumbens with spiperone, a dopamine antagonist, or the ventral tegmental area with gamma-amino butyric acid (GABA) produced dose-dependent reductions in water intake and number of laps taken while increasing the latency to drink. The spiperone injection did not alter the pressor response. On the other hand, the GABA injections attenuated the pressor responses to central angiotensin II administration. These findings suggest that GABA input to the ventral tegmental area modifies both the cardiovascular and drinking responses elicited following central administration of angiotensin II. However, the dopamine projections to the nucleus accumbens appear to be involved only in the drinking responses elicited by central injections of angiotensin II. Divergence for the coordination of the skeletal motor behavioral component and the cardiovascular component elicited by central administration of angiotensin II must occur before the involvement of these dopamine pathways.     

Thermogenic drinking: mediation by osmoreceptor and angiotensin II pathways

Exposure of rats to air at 5 C for 1-12 days is accompanied by a relative dehydration in spite of the continued presence of water. Dehydration during exposure to cold was manifested by: 1) a reduction in the ratio of water/food ingested; 2) an increase in the ratio of urine excreted/water ingested; 3) an increased evaporative water loss; 4) an increased serum osmolality and chloride concentration; and 5) a striking thirst and ingestion of water after transfer from cold to air at 26 C. Drinking began within 15 min and lasted approximately 1 h. Thermogenic drinking persisted for at least 120 days of exposure to cold. It was not thwarted by preventing access to water for either 1 or 2 h after transfer to warm air, but either intragastric or intraperitoneal administration of a water load equal to 3% of body weight inhibited water intake after transfer. These characteristics of thermogenic drinking are similar to those observed after 24 h of dehydration at 26 C; they also suggest that the cold-exposed rat is dehydrated relative to controls. These results suggest that osmoreceptors may play a role in the induction of thermogenic drinking. However, angiotensin II receptors may also play a role. Thermogenic drinking was inhibited by a beta 2-adrenergic, but not a beta 1-adrenergic, antagonist as well as by captopril, an inhibitor of the conversion of angiotensin I to angiotensin II. Further, plasma renin activity increased fourfold within 15 min after removal from cold. This suggests that an additional component involved in thermogenic drinking is the angiotensin II receptor. The extent to which thermogenic drinking is mediated by each pathway is unknown and will require additional studies.     

Angiotensin and other peptides in the control of water and sodium intake

Several neuroactive peptides have been implicated in thirst and sodium appetite in different species; three peptides are considered here. The best established of these is the octapeptide angiotensin II, which when administered systemically or intracranially causes completely normal drinking behaviour in all vertebrates tested, including many mammals, four or five birds, one reptile and one bony fish. In the rat, in which the original experiments were carried out, injection of a few femtomoles of angiotensin II caused a brisk drinking response within a minute or so of injection at a time of day when the animal would usually be resting. The response is usually completed within 10 min and after the larger doses the amounts of water taken may approach what the animal would normally drink in the course of 24 h. Another response to intracranial angiotensin, seen so far only in the rat, is an increase in sodium appetite. This is slower in onset than thirst, lasts for many hours and the response tends to become greater with repeated injections of hormone. Naturally occurring increases in sodium appetite may be caused by angiotensin generated by the action of cerebral isorenin. A second neuroactive peptide that affects thirst is the undecapeptide eledoisin, which is found in the salivary glands of certain Mediterranean cephalopods. Eledoisin and, to a lesser extent, substance P, with which it is related, are potent intracranial dipsogens in the pigeon, producing behaviour that is indistinguishable from that produced by angiotensin. However, in contrast to the stimulatory action of angiotensin on drinking behaviour in all other vertebrate species tested, these substances specifically depress drinking in the rat. A third peptide that has been implicated in thirst is antidiuretic hormone (ADH). This hormone has a profound but indirect effect on water intake in diabetes insipidus. In the dog, however, ADH in physiological amounts may influence thirst mechanisms by direct action on the central nervous system. In this species, but not in the rat, ADH lowers the threshold of thirst in response to osmotic stimulation and also to infusion of angiotensin. Of these three peptides, and others not mentioned here, angiotensin II has the best claim to be regarded as a neuroactive peptide. It alone is always dipsogenic when injected into the brain and it also stimulates sodium appetite. Whether the effects of angiotensin, on thirst and sodium appetite should be regarded as manifestations of the activity of a classical endocrine system, of a paracrine system, of a neurotransmitter system, or of all of these, cannot be decided at present. But these actions of angiotensin, when considered with its other actions on the distribution and conservation of body fluid, show that the hormone is intimately concerned in extracellular fluid volume control.     

Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Dipeptidyl-aminopeptidase III was isolated from cells of the cellular slime moldDictyostelium discoideum in the culmination stage of development. The enzyme was purified 18-fold by precipitation with ammonium sulfate and gel filtration chromatography and was shown to have a molecular weight of 158,000 and a sharp pH optimum at pH 10.2 and to be inhibited by sulfhydryl reagents. The enzyme acted upon the artificial substratearginyl-arginyl-β-naphthylamide, producing arginyl-arginine andβ-naphthylamine but notarginyl-β-naphthylamide. Activity towardarginyl-arginyl-β-naphthylamide was strongly inhibited by physiological concentrations of angiotensin III and, to a lesser extent, by angiotensins I and II and other angiotensin-related peptides but not by enkephalin peptides. Several dipeptides known to inhibit mammalian dipeptidyl-aminopeptidase III also inhibited theDictyostelium enzyme. Incubation of the enzyme preparation with angiotensins resulted in their conversion into a complex mixture of products. Thus dipeptidyl-aminopeptidase III fromDictyostelium closely resembles the mammalian enzyme in many of its characteristics.     

Central angiotensin II-induced pressor responses and neural activity in utero and hypothalamic angiotensin receptors in preterm ovine fetus

The central renin-angiotensin system is important in the control of blood pressure in the adult. However, few data exist about the in utero development of central angiotensin-mediated pressor responses. Our recent studies have shown that the application of ANG II into the fetal brain can increase blood pressure at near term. The present study determined fetal blood pressure and heart rate in response to a central application of ANG II in the chronically prepared preterm ovine fetus, determined the action sites marked by c-Fos expression in the fetal central pathways after intracerebroventricular injection of ANG II in utero, and determined angiotensin subtype 1 receptors in the fetal hypothalamus. Central injection of ANG II significantly increased fetal mean arterial pressure (MAP). Adjusted fetal MAP against amniotic pressure was also increased by ANG II. Fetal heart rate was subsequently decreased after the central administration of ANG II and/or the increase of blood pressure. ANG II induced c-Fos expression in the central putative cardiovascular area, the paraventricular nuclei in the brain sympathetic pathway. Application of ANG II also caused intense Fos immunoreactivity in the tractus solitarius nuclei in the hindbrain. In addition, intense angiotensin subtype 1 receptors were expressed in the hypothalamus at preterm. These data demonstrate that central ANG II-related pressor centers start to function as early as at preterm and suggest that the central angiotensin-related sympathetic pathway is likely intact in the control of blood pressure in utero.