Angiotensin II Type 1 (AT1) Receptor–Mediated Accumulation of Angiotensin II in Tissues and Its Intracellular Half-life In Vivo
Abstract Angiotensin II (Ang II) is internalized by various cell types via receptor-mediated endocytosis. Little is known about the kinetics of this process in the whole animal and about the half-life of intact Ang II after its internalization. We measured the levels of 125I–Ang II and 125I–Ang I that were reached in various tissues and blood plasma during infusions of these peptides into the left cardiac ventricle of pigs. Steady-state concentrations of 125I–Ang II in skeletal muscle, heart, kidney, and adrenal were 8% to 41%, 64% to 150%, 340% to 550%, and 680% to 2100%, respectively, of the 125I–Ang II concentration in arterial blood plasma (ranges of six experiments). The tissue concentrations of 125I–Ang I were less than 5% of the arterial plasma concentrations. 125I–Ang II accumulation seen in heart, kidney, and adrenal was almost completely blocked by a specific Ang II type 1 (AT1) receptor antagonist. Steady-state concentrations of 125I–Ang II were reached within 30 to 60 minutes in the tissues and within 5 minutes in blood plasma. The in vivo half-life of intact 125I–Ang II in heart, kidney, and adrenal was approximately 15 minutes, compared with 0.5 minute in the circulation. Thus, Ang II, but not Ang I, from the circulation is accumulated by some tissues, and this is mediated by AT1 receptors. The time course of this process and the long half-life of the accumulated Ang II support the contention that this Ang II has been internalized after its binding to the AT1 receptor, so that it is protected from rapid degradation by endothelial peptidases. The results of this study are in agreement with growing evidence of an important physiological role for internalized Ang II.
Receptor-mediated endocytosis of the vasoactive peptide angiotensin II (Ang II) is an important mechanism by which the in vivo activity of the renin-angiotensin system is regulated. Two pharmacologically distinct classes of cell surface receptors have been identified for Ang II, ie, type 1 and 2 (AT1 and AT2).1 Most of the classic physiological actions of the renin-angiotensin system appear to be mediated by the AT1 receptors.
Many cell surface receptors are internalized following binding to their agonists. Endocytosis of the complex of the AT1 receptor with its agonist Ang II has been demonstrated in a number of target cells, ie, vascular smooth muscle cells,2 3 renal tubule cells,4 and cells from the adrenal cortex and medulla.5 6 7 8 This provides a mechanism for regulating the number of receptors on the cell surface. Receptor-mediated endocytosis of Ang II and its subsequent degradation in lysosomes may also serve an important function in the disposal of this peptide.3 7
Although a plasma membrane localization is thought to be essential for Ang II receptor function, it has been suggested that internalization of the receptor is important for signal transduction. There is evidence that in cultured vascular smooth muscle cells, the delayed accumulation of the protein kinase C activator diacylglycerol in response to Ang II depends on receptor-mediated endocytosis of this peptide. An early step in this process seems to be important for the second and sustained phase of diacylglycerol accumulation.9 Receptor-mediated endocytosis of Ang II may also be important for the generation of inositol 1,4,5-triphosphate (IP3) and transport of sodium in response to stimulation of the apical AT1 receptors of proximal renal tubule cells.4 It has been reported that in adrenal glomerulosa cells, the inhibition of Ang II–induced internalization reduces the sustained phase of IP3 generation and abolishes the second phase of the cytoplasmic calcium response.10 11 It is known that stimulated steroidogenesis by these cells closely follows the changes in intracellular calcium.12 Others found that in adrenal glomerulosa cells, internalization of the AT1 receptor is required for protein kinase C activation but not for IP3 release and steroidogenesis.13
Evidence also suggests that selective intracellular delivery of internalized Ang II is necessary for an intracellular action. A high-affinity cytoplasmic Ang II binding protein with many characteristics of a receptor has been described.14 15 Ang II is rapidly accumulated in vascular and cardiac muscle cell nuclei,16 and AT1 receptor-like Ang II binding sites have been identified in liver cell nuclei.17 18 It has been reported that Ang II binds to chromatin and may influence transcriptional processes.19 20 21 Ang II induces the expression of proto-oncogenes and has growth-promoting effects in various cells.22 An intracellular action would require a sufficiently long half-life of internalized Ang II or a biologically active Ang II metabolite.
Previous studies of the cellular uptake and intracellular half-life of Ang II have been carried out in vitro. The in vitro studies made use of 125I-labeled Ang II but did not discriminate between intact 125I–Ang II and 125I-labeled peptide fragments. In the course of studies in our laboratory, aimed at quantifying Ang II production in different regional vascular beds, 125I-labeled Ang I or Ang II was infused into pigs.23 24 Here we report on the in vivo accumulation of intact 125I–Ang II in cardiac, renal, and adrenal tissues during these infusions and on the effect of a specific AT1 receptor antagonist on the Ang II uptake by these tissues. We compared the data on the tissue accumulation and half-life of 125I–Ang II with data obtained for 125I–Ang I. Unlike Ang II, Ang I is not biologically active and is probably not subjected to receptor-mediated endocytosis.
[Ile5]Ang-(1-10) decapeptide (Ang I), [Ile5]Ang-(1-8) octapeptide (Ang II), and [Ile5]Ang-(2-8) heptapeptide (Ang III) were obtained from Bachem. [Ile5]Ang-(2-10) nonapeptide was from Senn Chemicals. [Ile5]Ang-(3-8) hexapeptide [Ang-(3-8)], [Ile5]Ang-(4-8) pentapeptide [Ang-(4-8)], and [Ile5]Ang-(1-7) heptapeptide [Ang-(1-7)] were from Peninsula Laboratories. Mono-iodinated 125I–Ang I was prepared with the chloramine T method and purified as described previously.25 Mono-iodinated 125I-labeled preparations of Ang II, Ang III, Ang-(3-8), Ang-(4-8), Ang-(2-10), Ang-(1-7), and tyrosine were also made.25 The specific radioactivity of the 125I–Ang I and 125I–Ang II preparations was approximately 3.6×106 cpm/pmol.
Ang I and Ang II antisera prepared in New Zealand White rabbits were used to identify the peptides in high-performance liquid chromatography (HPLC) radioactivity peaks. Ang I antiserum cross-reacted with Ang-(2-10) (100%) but not (<0.1%) with Ang II, Ang III, Ang-(3-8), Ang-(4-8), or Ang-(1-7). Ang II antiserum cross-reacted with Ang III (55%), Ang-(3-8) (73%), and Ang-(4-8) (100%) but not (<0.2%) with Ang I, Ang-(2-10), or Ang-(1-7). These cross-reactivity patterns show that the antibodies in both antisera were directed against the C-terminal sequences of Ang I and Ang II.24
All experiments were performed under the regulations of the Animal Care Committee of Erasmus University, Rotterdam, Netherlands, in accordance with the “Guiding Principles in the Care and Use of Animals” as approved by the American Physiological Society. Twenty-three female pigs (crossbred Yorkshire×Landrace, Hedelse Varkens Combinatie, Hedel, Netherlands) with a body weight of 25 to 30 kg were included in the study. Some animals were also used for studies on the cardiac uptake of kidney-derived renin. For these studies, which extend our earlier observations of this subject26 and which will be reported in a separate article, renin release from the kidney was stimulated with the diuretic furosemide, 40 mg twice daily for 2 days before the experiments reported here. Other animals were pretreated with the angiotensin-converting enzyme inhibitor captopril, 25 mg twice daily for 2 days, for investigation of the effect of angiotensin-converting enzyme inhibition on the tissue levels of 125I–Ang I and 125I–Ang II.
Animals were sedated with 20 mg/kg ketamine IM (AUV) and anesthetized with 20 mg/kg sodium pentobarbital (Apharma) administered via a dorsal ear vein. They were intubated and connected to a ventilator for intermittent positive pressure ventilation with a mixture of oxygen and nitrogen (30%/70%). Respiratory rate and tidal volume were adjusted to keep arterial blood gases within the physiological range. For maintenance of adequate anesthesia, a 7F catheter was placed in the superior caval vein for administration of 8.5 to 10 mg/kg per hour sodium pentobarbital. Another 7F catheter was placed in the superior caval vein for infusion of saline to correct for fluid losses and administer the AT1 receptor antagonist L-158,80927 28 (a gift of Dr R.D. Smith, DuPont Merck Pharmaceutical Co). A 7F catheter was inserted, via the left carotid artery, into the left ventricle under radiographic control for infusion of 125I–Ang I or 125I–Ang II (see below). An 8F catheter was inserted into the descending aorta, via a femoral artery, for measurement of central aortic pressure and collection of arterial blood.
After administration of 4 mg pancuronium bromide (Organon Teknika), a midline thoracotomy was performed, and the heart was suspended in a pericardial cradle. An electromagnetic flow probe (Skalar) was placed around the ascending aorta for measurement of ascending aortic blood flow (cardiac output). After a stabilization period of 30 to 45 minutes following completion of instrumentation, baseline measurements of systemic hemodynamic variables were made, and blood samples were collected for determination of blood gases. The animals were then subjected to constant infusions of either 125I–Ang I or 125I–Ang II.
Infusions of 125I–Ang I or 125I–Ang II
125I–Ang I was infused into the left cardiac ventricle at a constant rate of approximately 5×106 cpm/min. Steady-state plasma levels of 125I–Ang I and 125I–Ang II were reached within 10 minutes.23 For determination of the time required for tissue levels to reach a steady state, heart, kidney, and adrenals were removed after various periods of 125I–Ang I infusion, ie, after 15, 60, or 120 minutes of 125I–Ang I infusion. Blood samples were taken from the aorta at 10 and 60 minutes of 125I–Ang I infusion.
In some experiments, in which 125I–Ang I had been infused for 15 minutes, heart, kidney, and adrenals were removed at 15 or 30 minutes after the infusion had been stopped, so that the in vivo tissue half-life of 125I–Ang I and II could be estimated. The in vivo half-life of 125I–Ang I and 125I–Ang II in the circulation was determined by measuring the plasma levels of 125I–Ang I and 125I–Ang II in blood samples taken from the aorta at 0.25, 0.5, 1, 1.5, and 2 minutes after the 125I–Ang I infusion had been stopped.
The effect of blockade of the AT1 receptor on the tissue levels of 125I–Ang I and 125I–Ang II during 125I–Ang I infusion was studied by administration of the AT1 receptor antagonist L-158,809, 1 mg/min IV for 10 minutes, 30 minutes before the start of the 125I–Ang I infusion. At this dose of the AT1 receptor antagonist, the pressor effect of systemically administered Ang II (0.1 to 1.0 μg/kg) is completely blocked.29 After 10 minutes of 125I–Ang I infusion, an arterial blood sample was taken, and heart, kidney, and adrenals were removed at 15 minutes of infusion.
125I–Ang II was infused into the left cardiac ventricle at a constant rate of approximately 3×106 cpm/min. Heart, kidney, and adrenal were removed at 15, 60, or 120 minutes of 125I–Ang II infusion. Blood samples were taken from the aorta at 10 and 60 minutes of 125I–Ang II infusion. A steady-state plasma level of 125I–Ang II had been reached by that time.30
Blood and Tissue Sampling
During 125I–Ang I and 125I–Ang II infusions, blood samples (10 mL) were taken from the aorta for measurement of the plasma levels of 125I–Ang I and 125I–Ang II. The blood was rapidly withdrawn with a plastic syringe containing the following inhibitors (0.5 mL inhibitor solution in 10 mL blood): 0.01 mmol/L remikiren (a gift of Dr W. Fischli, Hoffmann–La Roche), 6.25 mmol/L disodium EDTA, and 1.25 mmol/L 1,10-ortho-phenanthroline (Merck) (final concentrations in blood). It was then immediately transferred into prechilled polystyrene tubes and centrifuged at 3000g for 10 minutes at 4°C. Plasma was stored at −70°C and assayed within 3 days.
The heart was removed either immediately or at various times after 125I–Ang I or 125I–Ang II infusion had been stopped. Before its removal from the body, the heart was stopped by fibrillation while the radiolabeled peptide infusion was still running. Immediately after the heart had been removed, a piece of left ventricular free wall tissue (1 to 2 g) was excised and transferred into liquid nitrogen. The tissue was frozen within 15 seconds after the heart had been stopped. Subsequent to removal of the heart, the left kidney and both adrenal glands, and in some cases also part of the sternocleidomastoid muscle, were excised, and a piece of each tissue (0.5 to 1 g) was immediately transferred into liquid nitrogen. The piece of renal tissue was mainly renal cortex. These tissues were frozen within 60 seconds after the heart had been stopped. The frozen tissues were stored at −70°C and assayed within 3 days.
For study of the ex vivo degradation of 125I-labeled angiotensins in tissue, remaining parts of the left ventricular wall tissue, the kidney, and the adrenals were kept at 37°C. Pieces of tissue were then cut off and rapidly frozen in liquid nitrogen at various times (0 to 60 minutes) after the heart had been stopped. The frozen tissues were stored at −70°C and assayed within 3 days.
Measurements of 125I–Ang I and 125I–Ang II in Tissue and Plasma
Frozen tissue samples were homogenized with a polytron (PT10/35, Kinematica) in 20 mL ice-cold ethanol/0.1 mol/L HCl (4:1, vol/vol).26 Homogenates were centrifuged at 20 000g for 25 minutes at 4°C. Ethanol in the supernatant was evaporated under constant air flow. The remainder of the supernatant was diluted in 20 mL of 1% ortho-phosphoric acid and centrifuged again at 20 000g. The supernatant was diluted with an equal volume of 1% ortho-phosphoric acid and then concentrated by reversible adsorption to octadecylsilyl silica (Sep-Pak C18, Waters). Plasma was directly applied to Sep-Pak cartridges.
The Sep-Pak cartridges were conditioned with 5 mL methanol and equilibrated with 5 mL cold water. Samples were passed through the cartridges at 4°C, followed by a wash with 10 mL cold water. Adsorbed angiotensins were eluted with 2.5 mL of 90% methanol/10% water (vol/vol) into polypropylene tubes, and the eluted samples were dried under vacuum.
Separations were performed by reversed-phase HPLC using a Nucleosil C18 steel column of 250×4.6 mm and 10-μm particle size (Alltech) as previously described.25 Water for HPLC was prepared with a Milli-Q system (Waters). Mobile phase A was 25% methanol in 0.085% ortho-phosphoric acid, containing 0.02% sodium azide. Mobile phase B was 75% methanol in 0.085% ortho-phosphoric acid, containing 0.02% sodium azide. The flow was 1.5 mL/min, and the working temperature was 45°C. The vacuum-dried Sep-Pak extracts were dissolved in 100 μL of 0.085% ortho-phosphoric acid and injected. Elution was performed as follows: 85% A/15% B (vol/vol) from 0 to 5 minutes, followed by a linear gradient to 40% A/60% B (vol/vol) until 20 minutes. The eluate was collected in 20-second fractions into polystyrene tubes coated with bovine serum albumin (Sigma Chemical Co). The concentrations of 125I-labeled angiotensins and their metabolites in the HPLC fractions were measured in a gamma counter.
In previous studies, in which Ang I and II levels were measured in cardiac tissue, we added a known amount of 125I–Ang I as an internal standard before the extraction procedure. We then used the recovery of 125I–Ang I after HPLC separation to correct for losses (maximally 20% to 30%) occurring during extraction and separation. In the present study, we did not add 125I–Ang I as an internal standard because the tissue already contained 125I–labeled Ang I and metabolites. We therefore did not correct for losses of angiotensin during the extraction and separation procedures. This may have led to an underestimation (by maximally 20% to 30%) of the actual tissue levels.
Hemodynamic Effects of 125I–Ang I and 125I–Ang II Infusions
Baseline heart rate, cardiac output, and mean arterial pressure were similar in furosemide- and captopril-pretreated pigs and in pigs treated with the AT1 receptor blocker L-158,809 (Table⇓). 125I–Ang I and 125I–Ang II infusions did not affect any of these parameters (data not shown), in agreement with previous studies.23 30
Identification of 125I–Ang I, 125I–Ang II, and Their 125I-Labeled Metabolites in Blood Plasma and Tissue by HPLC
Satisfactory separations were obtained of 125I–Ang I and 125I–Ang II and of these peptides and most of their 125I-labeled metabolites (Fig 1⇓). A comparison of the retention times of the various 125I-labeled peptide standards demonstrated that the 125I–Ang I and 125I–Ang II peaks were virtually free of 125I–Ang III, 125I–Ang-(3-8), 125I–Ang-(4-8), 125I–Ang-(2-10), 125I–Ang-(1-7), and 125I-tyrosine.
In plasma, more than 90% of the radioactivity in the peak with the same retention time as 125I–Ang I was bound by Ang I antiserum, compared with less than 5% by Ang II antiserum. This peak was therefore identified as 125I–Ang I. No peak with this retention time was observed in skeletal muscle, and a very small peak with this retention time was sometimes observed in heart and kidney. Adrenal tissue showed a larger peak with a retention time similar to that of 125I–Ang I, but less than 10% of the radioactivity in this peak was bound to Ang I antiserum. This peak therefore was not 125I–Ang I.
In both plasma and tissues, more than 90% of the radioactivity in the peak with the same retention time as 125I–Ang II was bound by Ang II antiserum, compared with less than 3% by Ang I antiserum. This peak was therefore identified as 125I–Ang II.
In addition to the 125I–Ang I and 125I–Ang II peaks, separate peaks with retention times corresponding to 125I-tyrosine, 125I–Ang-(1-7), and 125I–Ang-(4-8) were observed in the tissues. More than 80% of the radioactivity in the peak with the retention time of 125I–Ang-(4-8) was bound by Ang II antiserum, whereas less than 10% was bound by Ang I antiserum. We therefore concluded that this peak was indeed 125I–Ang-(4-8). The radioactivity in the peaks with the retention times of 125I–tyrosine and 125I–Ang-(1-7) was not bound by these antisera. The conclusion that these peaks were indeed 125I-tyrosine and 125I–Ang-(1-7) needs further confirmation.
Accumulation of 125I–Ang II in Tissue and the Effect of AT1 Receptor Blockade
Fig 2⇓ shows the tissue levels of 125I–Ang II (expressed relative to 125I–Ang II plasma levels) after 60 minutes of 125I–Ang I or 125I–Ang II infusion. The 125I–Ang II levels in the kidney and adrenal (expressed per gram tissue) were 340% to 550% and 680% to 2100% of the 125I–Ang II level in plasma (expressed per milliliter plasma), respectively (ranges of six experiments). In the heart, the 125I–Ang II level was 64% to 150% of the level in plasma. The 125I–Ang II level in skeletal muscle was 8% to 41% of the level in plasma. Results obtained in the furosemide-pretreated pigs were similar to those in the captopril-pretreated pigs, and results obtained after 125I–Ang I infusion were similar to those obtained after 125I–Ang II infusion. In the kidney and adrenal, and also in the heart, the tissue levels of 125I–Ang II were too high to be explained by the presence of 125I–Ang II in the extracellular fluid. The tissue levels of 125I–Ang I were less than 5% of the plasma level.
These results demonstrate that Ang II from the circulation is accumulated by the heart, kidney, and adrenal and that in the case of the kidney and adrenal, the tissue concentration of the accumulated Ang II is several times the plasma concentration. Ang I from the circulation is not accumulated by these tissues.
Fig 3⇓ compares the 125I–Ang II tissue–to–blood plasma concentration ratios that were reached in heart, kidney, and adrenal after 60-minute infusions of 125I–Ang I or 125I–Ang II with the ratios after 15- or 120-minute infusions. It took between 30 and 60 minutes for 125I–Ang II to reach steady-state levels in the tissues. This is much longer than in plasma, where it takes fewer than 10 minutes to reach a steady state.23 25 30
The effect of the AT1 receptor antagonist L-158,809 on the accumulation of 125I–Ang II in heart, kidney, and adrenal was studied during a 15-minute infusion of 125I–Ang I. Skeletal muscle was not studied because of the apparent lack of 125I–Ang II accumulation in this tissue (see above). As shown in Fig 4⇓, L-158,809 caused nearly complete blockade of 125I–Ang II accumulation. L-158,809 had no effect on the plasma levels of 125I–Ang I and 125I–Ang II (Table⇑). Thus, the accumulation of Ang II from the circulation by heart, kidney, and adrenal appears to depend on AT1 receptors.
Ex Vivo and In Vivo Half-Life of 125I–Ang II in Tissue
The process of tissue removal, cutting, and transfer into liquid nitrogen took less than 1 minute. Still, 125I–Ang II metabolism might be so rapid that the measured levels of this peptide are substantially below the levels in vivo. We therefore investigated the decrease in the tissue levels of 125I–Ang II in heart, kidney, and adrenal while the pieces of tissue cut from these organs were kept at 37°C before they were transferred into liquid nitrogen. Skeletal muscle was not included in these experiments because of the apparent lack of 125I–Ang II accumulation in this tissue (see above).
As shown in Fig 5⇓, the ex vivo half-life of 125I–Ang II in heart, kidney, and adrenal was 30 minutes or longer. Thus, one can conclude that the 125I–Ang II levels that were measured in these tissues after they had been transferred into liquid nitrogen as quickly as possible were indeed representative of the levels in vivo.
To get some information on the in vivo half-life of 125I–Ang II in tissue, we measured 125I–Ang II levels in heart, kidney, and adrenal after these organs had been kept in the body for 15 or 30 minutes after the 125I–Ang I infusion had been stopped. As shown in Fig 6⇓, the tissue levels of 125I–Ang II 15 and 30 minutes after discontinuation of the 125I–Ang I infusion were 40% to 70% and 20% to 50% of the level immediately after discontinuation of the infusion (n=2). This corresponds with an in vivo half-life in tissue of approximately 15 minutes. The half-life of 125I–Ang II in the circulation was approximately 0.5 minute, in agreement with earlier studies.23 Thus, it appears that Ang II that is taken up from the circulation by the heart, kidney, and adrenal and is accumulated in these organs has a much longer half-life in tissue than in circulating plasma.
In the present experiments, we infused 125I–Ang I and 125I–Ang II at a constant rate into the left cardiac ventricle of pigs to investigate the uptake and degradation of Ang I and II in the tissues. The use of radiolabeled angiotensins is based on the assumption that the body does not distinguish between labeled and unlabeled peptides. In a previous study, also in pigs, arterial and venous plasma levels of 125I–Ang I and 125I–Ang II and unlabeled Ang I and II were measured in a number of regional vascular beds in animals receiving infusions of 125I–Ang I combined with unlabeled Ang I for comparison of the regional extraction rates of the arterially delivered labeled and unlabeled angiotensins.23 24 Ang I in that study was infused in quantities that were sufficiently high to ignore the levels of endogenous Ang I and II. The results showed little difference in regional extraction between labeled and unlabeled angiotensins. There was a difference in conversion rate, the conversion rate of 125I–Ang I to 125I–Ang II being two times that of Ang I to Ang II, but the degradation of 125I–Ang I into peptides other than 125I–Ang II occurred at the same rate as the degradation of Ang I. The 125I–Ang II degradation rate was not different from the Ang II degradation rate.30
Another important methodological aspect of our study is the possibility that the measured tissue levels of 125I–Ang I and II differed from the levels in vivo because of rapid degradation of these peptides after the 125I–Ang I or 125I–Ang II infusions had been stopped. We addressed this issue by investigating the ex vivo degradation of 125I–Ang I and 125I–Ang II when the tissues were kept at 37°C. On the basis of the results of these experiments, it can be concluded that the measured tissue levels of 125I–Ang II are probably representative of the levels present in vivo. The measured tissue level of 125I–Ang I was too low to permit any conclusion about its level in vivo.
The main new findings of the present study were (1) the time-dependent accumulation of arterially delivered intact Ang II in heart, kidney, and adrenal gland of the intact animal and the importance of AT1 receptors for this process, and (2) the long in vivo half-life of blood-derived intact Ang II in these tissues, as opposed to the short half-life of Ang II in the circulation.
The tissue levels of 125I–Ang II we measured were about half maximal after 15 to 30 minutes of 125I–Ang I or II infusion, and the maximum appeared to be reached within 1 hour of infusion. This time course of Ang II accumulation in the tissues is similar to that observed for the AT1 receptor–dependent accumulation of radioactivity in isolated bovine adrenocortical and chromaffin cells when incubated with 125I–Ang II.7 8 In these studies of isolated cells, surface-bound radioactivity was rapidly internalized. Within 15 minutes of incubation at 37°C, more than 50% of the total radioactivity of the adrenal cells was derived from internalized 125I–Ang II. In addition, the increase in the total specific (AT1 receptor–related) radioactivity of the cells during the first 30 minutes of incubation with 125I–Ang II represented the increase of the internalized fraction, the surface-bound fraction remaining more or less constant. Such rapid internalization was also observed in monolayer cultures of rat vascular smooth muscle cells2 3 and in COS-7 cells expressing the rat AT1 receptor.31 Also in our in vivo experiments, the progressive accumulation of 125I–Ang II in heart, kidney, and adrenal appeared to be mediated by AT1 receptors, since it was inhibited by the AT1 receptor antagonist L-158,809. Because of this and because of the similarities between the kinetics of the 125I–Ang II accumulation process in the intact animal and the kinetics of 125I–Ang II internalization by cells in culture, we conclude that the increase in the tissue levels of 125I–Ang II in heart, kidney, and adrenal that we observed during 125I–Ang I and 125I–Ang II infusion is mainly determined by AT1 receptor–mediated internalization and therefore reflects the increase in intracellular 125I–Ang II.
The steady-state tissue level of 125I–Ang II in the heart (expressed per gram tissue) was similar to that in plasma (expressed per milliliter plasma). In kidney and adrenal, the levels of 125I–Ang II in tissue were several times those in plasma. In contrast, the levels of 125I–Ang I in tissue were less than 5% of levels in plasma. These results illustrate that arterially delivered Ang I is not accumulated and probably not subjected to receptor-mediated internalization. The order of steady-state tissue–to–blood plasma 125I–Ang II concentration ratios, ie, adrenal>kidney>heart, is in agreement with the Ang II receptor densities in these organs.32 33 34 35 36 Since most of the 125I–Ang II in tissue seems to be localized in the cells after its internalization through AT1 receptors, it is indeed logical to assume that the tissue-to-plasma 125I–Ang II concentration ratio is proportional to AT1 receptor density. This ratio is not influenced by the level of endogenous Ang II to which the receptors are exposed, if the receptor occupancy is low. This may explain why the tissue-to-plasma 125I-Ang II concentration ratios after captopril treatment were similar to those after furosemide treatment.
Recently, Zou et al37 reported that [Val5]Ang II, during low-dose infusion of this peptide for 14 days in uninephrectomized rats, was accumulated by the kidney (the endogenous Ang II of the rat is [Ile5]Ang II). These authors suggested that renal accumulation of circulating Ang II might be caused by AT1 receptor–mediated endocytosis. In a subsequent study from the same laboratory,38 also in uninephrectomized rats, it was found that chronic low-dose infusion of [Ile5]Ang II increased the level of this peptide in the kidney and that this increase was prevented by the AT1 receptor antagonist losartan. From this, the authors concluded that chronic Ang II infusion leads to receptor-mediated internalization of Ang II, enhancement of intrarenal Ang II formation, or both. Our results indicate that the findings of Zou et al37 38 are indeed explained, at least in part, by AT1 receptor–mediated endocytosis.
There was little difference in the tissue–to–blood plasma concentration ratios of 125I–Ang II between the animals subjected to 125I–Ang I infusions after furosemide treatment and the animals subjected to 125I–Ang I infusions after captopril treatment. There was also little difference between the results obtained after 125I–Ang I infusion and those after 125I–Ang II infusion. This indicates that most of the 125I–Ang II in the tissues was derived from arterially delivered 125I–Ang II and not, via conversion, from arterially delivered 125I–Ang I.
An important difference between our experiments and the studies reported so far lies in the fact that we relied on measurements of the tissue levels of intact 125I–Ang II rather than total tissue radioactivity. This enabled us to provide an estimate of the in vivo intracellular half-life of intact 125I–Ang II by measuring its tissue levels at different times after 125I–Ang I infusion was discontinued. The intracellular half-life of 125I–Ang II (approximately 15 minutes) was much longer than its half-life in the circulation (0.5 minute). Endocytosis of Ang II may protect the peptide from rapid degradation by endothelial peptidases.
Activation of cell membrane–bound receptors is crucial for the physiological actions of Ang II. AT1 receptor antagonist drugs block this activation process, and it is generally believed that this mechanism underlies the beneficial effects of these drugs in hypertension and heart failure. However, these drugs also interfere with the receptor-mediated endocytosis of Ang II, and as our study demonstrates, the AT1 receptor antagonist L-158,809 reduces the tissue concentrations of blood-derived Ang II to very low levels. There is growing evidence that AT1 receptor–mediated endocytosis of Ang II is important for some physiological responses to Ang II.4 9 10 11 Our observations, which indicate that internalized Ang II has a much longer half-life than Ang II in the circulation, are in agreement with the concept that intracellular Ang II indeed has functional significance. This raises the possibility that reduced endocytosis of Ang II may contribute to the therapeutic effects of AT1 receptor antagonists.
This work was supported by the Netherlands Heart Foundation Research Grant 91.121 and the Dutch Kidney Foundation Research Grant 96.1585. We thank René de Bruin for his excellent technical assistance.
- Received November 4, 1996.
- Revision received December 3, 1996.
- Accepted December 18, 1996.
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