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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS
Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark, DK-5000, Odense, Denmark
Submitted 12 May 2003 ; accepted in final form 7 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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1/7) without affecting blood pressure, vasopressin, or glomerular filtration rate. The effects of ANG III exhibited complex dose-response relations. ANG IV and ANG-(1-7) were ineffective. It is concluded that 1) plasma clearances of ANG III and ANG IV are higher than those of ANG II; 2) ANG III is more potent than ANG II in eliciting immediate sodium and potassium retention, as well as aldosterone secretion, particularly at low concentrations; and 3) the complexity of the ANG III dose-response relationships provides indirect evidence that several effector mechanisms are involved. sodium excretion; antidiuresis; blood pressure; angiotensin peptides; metabolic clearance rate
First, studies of the actions of ANG III (des-Asp1-ANG II) in the rat (29, 32) and in anesthetized dogs (11, 24) indicate that infusion of ANG III might produce vasoconstriction, sodium retention, and aldosterone release. This response is thought to be similar to ANG II, although not as potent.
Second, the discovery of several putative receptors for various angiotensin peptides has generated interest in the actions of other peptides produced in vivo from ANG I, such as ANG IV (des-Asp1-Arg2-ANG II) and ANG-(1-7) (des-Phe8-ANG II). Recently, a novel AT4 receptor with high affinity toward ANG IV has been described in the rat kidney (27). Very little is known about the renal actions of ANG IV; so far the AT4 receptor seems mainly to be considered a neuroendocrine receptor (45), and, although the AT4 receptor has been found in the kidney, ANG IV-mediated renal effects remain obscure (27). Kohara et al. (34) and Ferrario et al. (22) showed that ANG-(1-7) is a biologically active circulating metabolite of the angiotensin peptide family, formed through pathways independent of converting enzyme, and that it possesses biological activity distinct from ANG II and ANG III. The data of DelliPizzi et al. (17) supported the hypothesis demonstrating a natriuretic response to ANG-(1-7) in the isolated rat kidney. Subsequently, several groups have shown an antidiuretic and antinatriuretic effect of ANG-(1-7) (23, 30), although in large doses given to anesthetized animals. ANG-(1-7) has also been shown to increase GFR, urine flow, and electrolyte excretion through production of prostaglandin I2 (31).
Third, characterization of the turnover rate of the various angiotensin peptides has been attempted in the conscious sheep model (11, 21), but not during blockade of the endogenous RAAS.
The present experiments were designed to study the metabolic clearance rate for the angiotensin fragments and to test the hypothesis that infusion of fragments of angiotensins, i.e., ANG III, ANG IV, and ANG-(1-7), during constant low-RAAS activity exerts biological effects, e.g., in blood pressure and in renal excretion rates of electrolytes and water, when infused into conscious dogs in amounts that can be considered physiological. Before the infusion of the angiotensin peptides, the RAAS was blocked acutely. This is a key feature of the present investigation. It is a substantial advantage to limit the endogenous peptide synthesis so that changes in endogenous production do not obscure the true effect of the infused peptides. Without angiotensin I-converting enzyme (ACE) inhibition, it would be impossible to determine the relative contribution of endogenous and exogenous peptide. Infused angiotensin would, to an unknown extent, replace a decrease in endogenous production induced by the elevated plasma peptide concentration. Furthermore, when studying the acute renal effects of angiotensin peptides, the effects of aldosterone should be inhibited to provide the best platform for observation of the specific peptide effects. This is, to our knowledge, the first study to use this approach when studying both turnover and physiological effects of different angiotensin peptides.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experiments were performed in six female conscious Beagle dogs weighing 12.2-15.1 kg. Experiments were approved by the Danish Animal Experiments Inspectorate. The dogs had free access to tap water and were fed a standard diet of commercial dog food (Maintenance, Hill's Pet Nutrition) consisting of one meal a day at 1430. Daily sodium intake was 2.2 ± 0.1 mmol/kg (mean ± SE, calculated from manufacturer-specific batch sodium analysis). Before the experiments, the dogs underwent two-stage surgery. The dogs were premedicated (propionylpromazine 0.15 mg/kg, methadone 0.25 mg/kg, atropine 0.02 mg/kg, and caprofen 40 mg/kg), and general anesthesia was induced by propofol (0.4 mg/kg) and maintained by inhalation of a mixture containing N2 and O2 (1:2) and 1-2% halothane. With the use of standard aseptic procedures, both common carotid arteries were displaced into skin loops to facilitate an arterial puncture. An episiotomy was performed during general anesthesia to ease catheterization of the bladder. After at least 3 wk, a bilateral ovariectomy and hysterectomy were performed to eliminate possible interference by gonadal hormones. The dogs were trained to stand quietly, supported by a canvas sling, throughout the entire experiment (5 h).
Experimental Protocol
Each dog participated in all experiments, with intervals of at least 14 days. At midnight before the day of experiment, an electric valve, controlled by a timer, interrupted the water supply. The dogs were brought into the laboratory at 0630, and 3 h before the first measurements they were given an intravenous bolus injection of potassium canrenoate, a water-soluble aldosterone antagonist (Soldactone, 6 mg/kg, Searle Scandinavia, Malmö, Sweden). At 0830, a sterile catheter (Intracath, Becton Dickenson, Sandy, UT) was introduced into a jugular vein, and the tip was placed in the right atrial area and used for infusions. Another catheter (Vasculon, Becton Dickenson) was inserted into the common carotid artery for continuous measurements of blood pressure and sampling of arterial blood. A silicone Foley catheter (Argyle, Sherwood Medical, Tullamore, UK) was introduced into the bladder. GFR was estimated by using clearance of exogenous creatinine. One hour before data sampling, an intravenous bolus of creatinine (8.2 ml 
14 mg/kg) was given, followed by a continuous infusion of creatinine (7.2 ml/h 12.5 mg·kg-1·h-1). Simultaneously, a bolus of converting enzyme inhibitor enalaprilate maleate (2 mg/kg; Sigma, St. Louis, MO) was given intravenously, and a continuous infusion of canrenoate (1 mg·kg-1·h-1) was initiated. The latter was continued throughout the experiment. At 1000 (time = 0 min), the experiment was initiated. After a 30-min control period, infusion of vehicle or synthetic peptide [ANG II, ANG III, ANG IV, or ANG-(1-7)] was initiated and continued for 120 min. The infusion of peptide was followed by a 30-min recovery period. The time line of the experiments is shown in Fig. 1.
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Throughout the 3-h experiment, urine was collected every 30 min. At the end of each period, the bladder was flushed three times with distilled water, with careful avoidance of air in the drainage system. Urine samples were obtained for the determination of osmolality, sodium, potassium, and creatinine. Small samples of arterial blood (1 ml) were obtained at the beginning of the experiment and at the end of each sampling period for determination of plasma creatinine. Larger samples (17 ml) were collected at time = 25, 85, 145, and 175 min for analyses of electrolyte, protein, and hormone concentrations (see Fig. 1).
All peptides were purchased from Peninsula Laboratories. Each peptide was first dissolved in aqueous acetic acid (0.25% adjusted to pH = 4.5) to a 10 nmol/ml stock solution stored at -18°C. The stock solution was further diluted in a hypotonic solution of glucose and urea to a 600 pmol/ml working solution immediately before infusion.
In all series, peptides were administered at a rate of 6.0 pmol·kg-1·min-1, except 1) in additional separate experiments where ANG III was infused at 30 pmol·kg-1·min-1 for 1 h and then at 150 pmol·kg-1·min-1 in the subsequent hour and 2) in time control experiments in which only vehicle was infused.
Hemodynamics
A pressure transducer (BLPR, World Precision Instrument, Hertfordshire, UK) measured the arterial blood pressure continuously. The signal was amplified (preamplifier, PB1-C, World Precision Instruments) and digitized by a computer at a frequency of 100 Hz by using a standard IO card (National Instruments, Austin, TX) and custom-designed software (Lab View, National Instruments). Beat-to-beat systolic, diastolic, and mean pressures and heart rate were determined from the pressure curve. Data were stored on disk and averaged over 30-min periods.
Analyses
Plasma and urine osmolality were measured by freezing-point depression (model 3D3, Advanced Instruments, Needham Heights, MA). Sodium and potassium ion concentrations were measured by flame photometry (model IL 243, Instrumentation Laboratory, Lexington, MA), and plasma protein concentrations were measured by refractometry (refractometer model T2-Ne, Atago, Tokyo, Japan). Plasma and urine concentrations of creatinine were determined by a creatinine autoanalyzer (Creatinine analyzer 2, Beckmann, Fullerton, CA).
Plasma Hormones
Arterial blood samples for hormone analysis were obtained in prechilled 10-ml polyethylene tubes (Minisorb, Nunc, Denmark) containing EDTA (25 µmol) and aprotinine (NovoNor-disk, Bagsvaerd, Denmark). The samples were centrifuged at 4°C for 10 min and then stored at -18°C until extraction. Hormone extraction was performed according to the method described by Plovsing et al. (38).
Vasopressin. Plasma vasopressin was measured by using an antibody (AB3096) produced in this laboratory and by the methods described by Emmeluth et al. (19). Detection limit was 0.16 pg/ml of plasma, and mean recovery of unlabeled vasopressin was close to 70%. Intra- and interassay coefficients were 4.9 and 7.3%, respectively.
ANG immunoreactivity. The plasma ANG immunoreactivity (ANG IR) was measured in extracts of plasma by using an antibody (Ab-5-030682). Separate measurements demonstrated 100% cross-reactivity of ANG III and ANG IV, but the antibody does not recognize either ANG I or ANG-(1-7) (cross-reactivity <10-4). Detection limit was 1.0 pg/ml of plasma, and mean recoveries of unlabeled peptide added to dog plasma for ANG II, ANG III, and ANG IV were 91, 76, and 93%, respectively. Assuming stable concentrations in plasma of endogenous peptides during peptide infusion, the increase in ANG IR can be used to determine metabolic clearance rates of the various peptides. For these calculations, all values were corrected for incomplete recovery determined in each individual assay.
Aldosterone. Plasma aldosterone was measured without extraction by using a commercial kit (Coat-A-Count, Diagnostic Products, Los Angeles, CA). Detection limit was 11 pg/ml, and intra- and interassay coefficients of variation were less than 2.5 and 5.3%, respectively.
Atrial natriuretic peptide. Plasma atrial natriuretic peptide (ANP) was measured according to methods previously described by Schütten et al. (41) with minor modifications. Extraction recovery was 73%. Intra- and interassay variations were <4.3 and 4.8%, respectively, and detection limit was 2.0 pg/ml.
Statistics
The data are presented as means ± SE. Asterisks indicate values different from preinfusion period. Daggers indicate values different from time control series. Results were evaluated by a one-way ANOVA for repeated measurements between and within groups. If the results of the ANOVA were significant (P < 0.05), all differences were investigated by Newman-Keuls test. P < 0.05 as considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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During time control experiments, plasma ANG IR was low and constant at 5 pmol/l (baseline, Fig. 2). The infusion of ANG II raised ANG IR by 60 pmol/l. This elevation was stable throughout the infusion and corresponds to a metabolic clearance rate of ANG II of 100 ml·kg-1·min-1 (Table 1). Administration of equimolar amounts of ANG III generated increases in ANG IR much smaller than those of ANG II. In addition, infusion of ANG III did not produce a steady-state situation as seen in the ANG II series. The ANG III infusion raised ANG IR to 30 pmol/l above baseline after 1 h of infusion, but it declined significantly to 15 pmol/l in the subsequent hour, despite the ongoing constant infusion. Despite the lack of a steady-state situation, clearance values were calculated at the end of both the first and the second hour of infusion. These values must be regarded as minimal clearance rates. The ANG IV infusion produced a constant elevation similar to that of the second hour of ANG III infusion, so that mean ANG IR was 16 pmol/l throughout the ANG IV infusion.
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Metabolic Clearance Rate
Calculated for each hour of infusion, the clearances of ANG II were similar (Table 1). The clearances of ANG III and ANG IV were significantly higher (by factors 2 and 4, see Table 1). The clearance for ANG III was not significantly different from the clearance of ANG II in the first calculation period, but the subsequent increase in ANG III clearance was highly significant. The clearance rates of ANG IV were similar in the two calculation periods. In the experiments with infusion of larger amounts of ANG III, the metabolic clearance rate declined in the second period.
Cardiovascular Variables
After ACE inhibition, mean arterial blood pressure (MABP) was 97 mmHg (Table 2). With ANG II infusion, it increased to 120 mmHg, returning gradually to the preinfusion level in the recovery period, for which the 30-min mean was 106 mmHg. At the end of the experiments, blood pressure was similar to preinfusion level. Heart rate remained unchanged.
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ANG III (6 pmol·kg-1·min-1) produced a tendency toward an increase in blood pressure (from 96 to 106 mmHg, P 0.055). With the larger doses of ANG III, the dogs became markedly hypertensive. At 30 pmol·kg-1·min-1, blood pressure rose almost instantly to a mean of 123 mmHg, i.e., very similar to that generated by ANG II at 6 pmol·kg-1·min-1. A further increase was observed when the infusion rate was increased to 150 pmol·kg-1·min-1; in the first 30-min period, a mean value of 140 mmHg was obtained, reflecting a gradual increase in this period to the blood pressure of 153 mmHg, which remained stable in the last 30-min infusion period (Table 2). In the recovery period, blood pressure returned almost immediately to its control value (101 mmHg). ANG IV and ANG-(1-7) did not cause significant changes in blood pressure. Low- and constant-control values of heart rate, 70 beats/min, were observed in all series. Heart rate changed significantly, but modestly, in individual periods of some of the series; however, the changes appeared to be unrelated to infusions.
Plasma Variables
Only minor changes were observed in plasma osmolality, plasma protein, plasma sodium, and plasma potassium (Table 3).
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Vasopressin
Although plasma osmolality tended to decline throughout the experiments, the plasma vasopressin concentration almost doubled (+88%) on infusion of ANG II (Table 3). However, this plasma level was not sustained throughout the 2-h infusion period, despite a constant high ANG IR. In the ANG-(1-7) series, the plasma concentration of vasopressin was low and constant, as in the time control series. Within the ANG IV series, the plasma vasopressin level increased modestly, but to values lower than in time control experiments. In contrast, the larger dose of ANG III stimulated vasopressin release dose dependently.
Aldosterone
In the time control series, plasma aldosterone was declining throughout the experiment (Table 3). Infusion of ANG II and ANG III stimulated aldosterone release similarly (plasma concentration rose to 345 ± 72 and 268 ± 71 pg/ml, respectively). Surprisingly, the high dose of 30 pmol·kg-1·min-1 ANG III did not increase plasma aldosterone more than infusion of 6 pmol·kg-1·min-1. Further elevation of the ANG III dose to 150 pmol·kg-1·min-1 did indeed increase aldosterone concentration to 550 pg/ml. Neither ANG IV nor ANG-(1-7) raised plasma aldosterone concentration. In contrast, declines similar to time control experiments were observed in both cases.
ANP
The ANP concentration also declined steadily through the time control series. During infusion of both ANG II and ANG III, the plasma ANP concentration increased without a concomitant increase in the sodium excretion. In the ANG IV and ANG-(1-7) series, the plasma ANP concentration declined in a manner very similar to that in time control experiments.
Renal Variables
ANG II. ANG II markedly reduced GFR, which decreased by some 18% with the onset of peptide infusion (Table 4). Urine flow immediately decreased to about one-third of preinfusion value, as did sodium excretion. However, in the recovery period, the sodium excretion continued to be low, at 20% of preinfusion value, and surprisingly no rebound effect was observed. Urine osmolality increased during ANG II infusion (Table 4).
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ANG III. ANG III at the standard dose of 6 pmol·kg-1·min-1 did not affect GFR significantly, although a tendency toward a reduction was seen. A significant decrease in GFR was produced (from 38 to 27 ml/min) by the higher ANG III dose of 30 pmol·kg-1·min-1 (Table 4). The highest dose of 150 pmol·kg-1·min-1 did not reduce GFR further. The initial decrease in sodium excretion was very similar during equimolar infusions of ANG II and ANG III (Fig. 3), despite the large difference in ANG IR. In the recovery period, an immediate, remarkable rebound was observed in sodium excretion as well as urine flow. The fivefold higher dose of ANG III (30 pmol·kg-1·min-1) lowered sodium excretion to a minimum of 7 µmol/min. The increase to 150 pmol·kg-1·min-1 failed to produce any additional antinatriuretic response. After the infusion, sodium excretion immediately returned toward control levels, in contrast to the persistent antinatriuresis observed after ANG II infusion. Urine osmolality was unaffected by the low (6 pmol·kg-1·min-1) dose of ANG III, but increased to reach statistical significance on administering higher doses (30 and 150 pmol·kg-1·min-1).
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ANG IV and ANG-(1-7) series. GFR did not change on infusion of either of these peptides. Mean sodium excretion and urine flow were not significantly different from time control levels in either series.
Time control series. All renal variables remained stable throughout the time control series.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The pretreatment was performed acutely before each experiment to avoid any significant changes in body fluid status, and the applied regime was very effective in producing stabile natriuresis and diuresis (Table 4 and Fig. 3), as seen by Bie et al. (7) and Emmeluth et al. (19). The ANG IR, however, did not become immeasurable on ACE inhibition. Nussberger et al. (37) showed that ANG II becomes virtually undetectable during enalaprilate treatment, and hence the immunoreactivity picked up by the RIA could well be cross-reacting peptides as well as angiotensins formed by pathways not involving ACE. Nevertheless, the low and stable ANG IR and constancy of renal function in the time control experiments demonstrate that a very stable platform was used to test the effects of the angiotensin peptides.
Metabolic clearance rate during blockade of endogenous RAAS, along with renal and cardiovascular measurements, has, to our knowledge, not been reported previously. Other groups have measured metabolic clearance rate or turnover of ANG II and related peptides, but not under the similar strictly standardized conditions. Fei et al. (21) found the metabolic clearance rate for ANG II (103 ± 16 l/h) and ANG III (125 ± 27 l/h) to be significantly different. Britton et al. (11) argued that, compared with ANG II, more ANG III was needed to produce a given decrease in renal blood flow. This would be compatible with a higher turnover rate of ANG III compared with ANG II. Vernace et al. (44) showed that the metabolic clearance rate of ANG II increased on AT2 receptor inhibition by PD-13319. It is possible, but unproven, that a coupling exists between the AT2 receptor and the mechanisms by which ANG II is degraded, possibly by an intracellular pathway. This is not completely unlikely because the intracellular response of the AT2 receptor remains obscure. If AT2-receptor occupancy is normally associated with inhibition of degradation mechanisms, this would explain that the clearance of ANG II was increased by AT2-receptor blockade. The increased clearance rates of ANG III and ANG IV in our study could be explained by such a phenomenon, provided that the binding affinities between ANG III and ANG IV and the AT2 receptor are different from that of ANG II. The AT2 receptor may in this way act as a clearance receptor, although not in the classic sense where the clearance from the circulation takes place by receptor-mediated endocytosis. The phenomenon of endocytotic clearance receptors is known from other receptor systems, including ANP, glycoprotein, and lipoprotein clearance receptors (1, 4, 25). The receptor responsible for the greater turnover of ANG III and ANG IV compared with ANG II could also be a non-AT1 and non-AT2 receptor. Most likely plasma angiotensinases (N- or A-aminopeptidase) are not responsible for the faster turnover, because the activity of these enzymes is relatively moderate as plasma aminopeptidases have been shown to clear angiotensins from plasma at a disappearance rate corresponding to a half-time of 30 min (33). Therefore, the angiotensinases circulating in plasma do not seem to be important in degrading the angiotensins, in contrast to tissue angiotensinases and membrane-bound receptor systems.
Our data show very clearly that both ANG III and ANG IV are metabolized much faster than ANG II (see Table 1). Although the ANG III turnover was not calculated from steady-state values, they represent the minimal clearance rates of ANG III. The true clearance rate may be much higher than these calculated values. The differences mentioned above between the present results and the data obtained by Fei et al. (21) and Britton et al. (11) might be attributable to differences in experimental design. In our study, the endogenous RAAS was inhibited pharmacologically, in contrast to the other two studies. Our data, therefore, are not influenced by possible changes in endogenous generation rate of ANG II during infusion of the peptide. In addition, the calculations of Fei et al. (21) are based on arteriovenous measurements, whereas our calculations are based on continuous infusions to a steady-state level of peptide. Furthermore, a substantially larger clearance of ANG III compared with ANG II is compatible with the rapid return of renal and cardiovascular variables toward control level, which was seen in the recovery period. ANG III infusions (both 6 and 150 pmol·kg-1·min-1) showed a remarkably rapid off-effect compared with ANG II; after infusion of the latter, GFR, urine flow, and sodium excretion showed no sign of rebound in the recovery period, despite the finding that the ANG IR in plasma returned to preinfusion level. Our results demonstrate that it is essential to measure plasma concentrations of the various peptides to evaluate their potency. Due to marked differences in plasma clearance of the peptides, it is invalid to assume that similar plasma concentrations are generated by equimolar infusion rates.
Larger clearance rates of ANG III and ANG IV compared with ANG II were also found in investigations performed on healthy humans in our laboratory (38), where infusion of ANG III elicited substantial effects on aldosterone secretion and renal function at doses too low to generate measurable increases in ANG IR.
The observed effects of the ANG II infusion were expected (20, 26). ANG IR showed a steady state of 65 pmol/l, and this led to a decrease in sodium excretion, GFR, and urine flow, and an increase in MABP and urine osmolality. Heart rate and plasma electrolyte, osmolality, and protein concentration were unaffected by peptide infusion. Constancy of heart rate is a well-known phenomenon during ANG II-mediated changes in blood pressure (8). Usually, the actions of ANG II are immediate and related to the plasma concentration of the peptide. Therefore, it is puzzling that sodium excretion, GFR, and urine volume remained depressed in the recovery period under conditions in which ANG IR and blood pressure had returned almost to preinfusion levels.
The effects of ANG III were surprising in the sense that a much lower plasma concentration was needed to produce a very potent antinatriuretic response immediately after the onset of the infusion compared with ANG II. The antinatriuretic effect was observed without changes in blood pressure, vasopressin, or GFR. This supports the findings made by Huang (32), who reported that ANG III produced antinatriuresis without an increase in blood pressure. However, the present finding of high potency of ANG III over that of ANG II has not been reported previously. Furthermore, the increase in blood pressure produced by ANG II does not lead to pressure natriuresis, because a concomitant reduction in GFR is observed. Therefore, pressure natriuresis cannot explain that, relative to ANG II, a much lower plasma concentration of ANG III was needed to produce the same reduction in sodium excretion. Freeman et al. (24) showed a tendency toward a reduction in sodium excretion in anesthetized dogs on ANG III infusion at 25 ng·kg-1·min-1 (corresponding to 23 pmol·kg-1·min-1) without significant changes in GFR or MABP. Using approximately the same dose of ANG III (30 pmol·kg-1·min-1), we find a more potent response in our experiments, including a 27% reduction in GFR (37 ± 2 to 27 ± 2 ml/min), along with an 85-90% decrease in sodium excretion (57 ± 8 to 7 ± 1 mol/min) and urine flow (0.9 ± 0.3 to 0.1 ± 0.0 ml/min). This clearly demonstrates that, when the plasma concentration of ANG III is brought to a level comparable to that of ANG II, the effects of ANG III are in some respects quite similar (GFR, increases in urine osmolality, plasma AVP, plasma aldosterone) and, in others, markedly exaggerated (Na+, K+ retention). We observed that, at similar levels of ANG IR, the blood pressure elevations generated by ANG II and ANG III were identical (to 121 ± 4 and 123 ± 3 mmHg, respectively). This supports the notion that ANG II and ANG III bind to the same receptors in the vasculature with similar affinities, probably to the AT1 receptor, as reported by Chiu et al. (15). However, the antinatriuretic response to ANG III infusion may, in fact, be mediated through a mechanism entirely different from that activated by ANG II.
Several groups have shown previously that ANG III stimulates aldosterone secretion in a manner similar to ANG II (9, 36); however, the relative contribution of the peptides to aldosterone release has remained obscure. Chiu and Peach (16) reported equal stimulation of aldosterone synthesis in cell suspensions stimulated by 10-7 M concentrations of ANG II and ANG III. Studies performed in pentobarbital-anesthetized rabbits (40) indicated very similar aldosterone response on ANG II and ANG III stimulation. Furthermore, Campbell et al. (13) showed, in conscious rats, that ANG II and ANG III in supraphysiological doses (100 ng/kg) produced similar effects on aldosterone release. Years ago, Bean et al. (5) demonstrated a linear relation between plasma ANG II, in the range of 20-100 pmol/ml, and plasma aldosterone concentration in the conscious Beagle dog. This could support the hypothesis that the mechanism or mechanisms, by which aldosterone secretion is stimulated by ANG II and ANG III, most likely are not identical.
When two peptides are compared on the basis of identical infusion rates, the plasma clearances must be assumed to be equal to interpret the data in a useful manner. In the present study, we demonstrate that ANG III is significantly more potent than ANG II with regard to aldosterone release, when estimated on the basis of plasma concentrations of ANG II and ANG III (Table 3). This is also true in healthy humans (38), in whom ANG III increases plasma aldosterone concentrations on ANG III infusion at such a low rate that plasma ANG III does not rise measurably. In the present study, aldosterone release was remarkably sensitive to small amounts of ANG III, but surprisingly insensitive to increases in plasma ANG III concentration, as identical aldosterone responses were observed during both 6 and 30 pmol·kg-1·min-1 infusion rates. Semple and coworkers (42) demonstrated higher potency of ANG II compared with ANG III in eliciting an increase in plasma aldosterone concentration. Furthermore, ANG III may not be essential to the aldosterone release mechanism; Douglas et al. (18) showed that ANG II is a potent stimulator of aldosterone without prior conversion to the heptapeptide. These observations do not correspond to our findings. However, differences between experimental protocols, e.g., endogenous RAAS blockade, may again be the reason. We find a nonlinear relation between plasma ANG III concentration and aldosterone release, which might be explained by operation of two receptor mechanisms: one with a high affinity and low capacity, possibly triggered only by ANG III, and another with a lower affinity, but a larger maximal response.
ANG-(1-7) administered in a low dose did not change the measured renal and cardiovascular variables. When ACE is inhibited, more substrate is available for the ANG-(1-7) producing pathway. ACE is a key enzyme in the bradykinin turnover; consequently, ACE inhibition will increase the circulating bradykinin and nitric oxide formation. Effects of ANG-(1-7) might be blunted by the ACE inhibition, which is reported to increase the bradykinin concentration by a factor of 8, because ANG-(1-7) is thought to work partly through an increase in bradykinin concentration due to competition for ACE (12, 46). Other investigators (23, 27, 30) have shown both diuretic and natriuretic effects of circulating ANG-(1-7), but in doses much greater than used in the present study.
In the present setting, absence of an effect is not particularly informative, especially as ANG IV and ANG-(1-7) may play important local and paracrine roles in the organism and may even be important as classic hormones in amounts larger than those studied here.
Perspectives
The present study provides further evidence that the effect of the RAAS is more complex than previously believed. ANG III seems to be a more important mediator of aldosterone secretion and sodium excretion than previously thought. Our results include patterns of effects, which are very difficult to explain based on known receptor systems. Therefore, they provide evidence, albeit indirect, that all types of receptors sensitive to angiotensin peptides might not yet have been identified. It would be interesting to elucidate the exact mechanism by which ANG III is working. The present investigation was not designed to do so. Nevertheless, it is tempting to suggest that a plausible explanation of our data requires a very sensitive and specific ANG III receptor in addition to ANG III actions elicited via ANG II receptors. Should a pathway different from the well-known ANG II receptor pathways be found to mediate some of the effects of ANG III, particularly at low concentrations, it could be an important element in the normal physiological homeostasis, as well as in the pathophysiology of major diseases, e.g., hyperaldosteronism and essential hypertension.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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H. R. Kirchheim Our fragmentary knowledge of the regulatory functions of ANG II "fragments": are we beginning to see the light? Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2003; 285(5): R937 - R938. [Full Text] [PDF]
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R. R. Plovsing, C. Wamberg, N. C. F. Sandgaard, J. A. Simonsen, N.-H. Holstein-Rathlou, P. F. Hoilund-Carlsen, and P. Bie Effects of truncated angiotensins in humans after double blockade of the renin system Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2003; 285(5): R981 - R991. [Abstract] [Full Text] [PDF]
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, ANG II series; 
, ANG III series; 
, ANG IV series; 
, ANG III high-dose series. Synthetic peptide was infused at 6 pmol·kg-1·min-1 from time (t) = 30-150 min in all series except time control and ANG III high dose (30 pmol·kg-1·min-1 from t = 30-90 and at 150 pmol·kg-1·min-1 from t = 90-150 min). Values are means ± SE. *Significantly different (P < 0.05) from control value (0-30 min).
, ANG-(1-7) series; 
Significantly different (P < 0.05) from control series.