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Endocrinology Vol. 138, No. 2 725-733
Copyright © 1997 by The Endocrine Society

Articles

Angiotensin II Receptor Subtypes AT1 and AT2 Are Down-Regulated by Angiotensin II through AT1 Receptor by Different Mechanisms1

Rachida Ouali, Marie-Claude Berthelon, Martine Bégeot and José M. Saez

INSERM-INRA U-418, Hôpital Debrousse, Lyon, France

Address all correspondence and requests for reprints to: Dr. J. M. Saez, INSERM-INRA U-418, Hôpital Debrousse, 69322 Lyon Cedex 05, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The regulatory effects of angiotensin II (AngII) on its receptor subtypes, AT1 and AT2, were studied using cultured bovine adrenal cells (BAC), which express both receptor subtypes, and PC12W and R3T3 cells, which express only AT2 receptors. In BAC, AngII caused a decrease in AT1- and AT2-binding sites and their corresponding messenger RNAs (mRNAs), but with different kinetics. AT1-binding sites decreased by more than 50% within the first 3 h, whereas AT1 mRNA started to decline after a lag period of 3 h. Both AT2-binding sites and mRNA remained stable within the first 6 h of AngII treatment. Then, AT2 mRNA decreased rapidly with an apparent half-life of 2–3 h, whereas AT2-binding sites declined with an apparent half-life of about 16 h. Measurement of transcription rate and mRNA half-life by the [3H]uridine-thiouridine method revealed that AngII reduced by 90% the rate of AT1 transcription, but had no effect on AT1 mRNA half-life, whereas it slightly reduced AT2 transcription, but markedly reduced AT2 mRNA stability. All of the effects of AngII on both AT1 and AT2 receptors were blocked by losartan, indicating that they were mediated exclusively through the AT1 receptor. In PC12W cells, AngII was unable to modify AT2-binding sites or mRNA. Moreover, in BAC, [125I]AngII was internalized through the AT1 receptor, whereas occupancy of AT2 receptors in either BAC or PC12W did not produce internalization of the hormone. These results indicate that AngII, through the AT1 receptor, down-regulates both AT1 and AT2, but by different mechanisms; AT1 receptor is regulated through internalization-degradation of the occupied receptor and inhibition of transcription, whereas AT2 receptor is regulated mainly by decreasing the stability of its mRNA. Moreover, the phorbol ester phorbol 12-myristate 13-acetate mimicked most of the effects of AngII in BAC and decreased both AT2-binding sites and mRNA on PC12W cells, indicating that the hormonal regulation of both AT1 and AT2 receptors is mediated through protein kinase C activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE hormone angiotensin II (AngII) exerts a variety of actions in the brain, adrenal gland, and kidney (1). Recently, two AngII receptor subtypes, named AT1 and AT2, were characterized in peripheral tissues and the brain on the basis of their sensitivity to sulfhydryl reducing agents and selective AngII receptor antagonists (2). AT1 receptor is sensitive to sulfhydryl reducing agents and has a high affinity for losartan or DUP753, whereas AT2 receptor is insensitive to sulfhydryl reducing agents and has a high affinity for the nonpeptide PD123319 and the peptide CGP42112A. Both AT1 (3, 4) and AT2 (5, 6) receptors have been cloned and shown to have the structural features of a putative seven-transmembrane domain receptor.

Several in vitro studies have shown by both radioligand-binding techniques (7, 8, 9, 10) and electron microscopy (11) that binding of AngII to its receptor initiates its internalization and sequestration. As only about 25% of the internalized receptors are recycled to the plasma membrane (10), it has been suggested that degradation of the internalized receptor is the main mechanism by which AngII regulates its own receptor. However, as in most of the above studies the total number of AngII-binding sites was measured, the effect of the hormone on AT1 and AT2 receptors was not possible to determine. Recent studies indicate that in several cell types AngII is also able after several hours to decrease AT1 messenger RNA (mRNA) levels (12, 13, 14, 15), but the effects in vivo are less clear (16, 17, 18). Studies on the regulation of AT2 receptor by AngII have been limited to studies of binding sites in two models with contradictory results. In cultured rat ovarian granulosa cells, which only express AT2 sites (19), AngII binding was down-regulated by cAMP derivatives, serum, and AngII (20). In R3T3 cells, which selectively express AT2 receptors, they appear to be down-regulated by growth factors, but are increased by AngII and nonpeptide- and peptide AT2-selective antagonists (21).

In the present study, using two cell types, one expressing both receptor subtypes and the other expressing only AT2 receptor, we address the following question. 1) What are the effects of AngII on AT1- and AT2-binding sites and mRNA? 2) Which receptor subtype is involved? 3) By which mechanism, transcriptional or posttranscriptional, does AngII regulate both receptor subtypes?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic AngII was obtained from Bachem (Bubendorf, Switzerland). Ham’s F-12 medium-DMEM (DMEM/F12), nystatin, penicillin/streptomycin, and FCS were obtained from Life Technologies (Paris, France). Bacitracin, insulin, transferrin, and phorbol ester, phorbol 12-myristate 13-acetate (PMA), were purchased from Sigma Chemical Co. (St. Louis, MO). The nonpeptide antagonists losartan (DUP753) and PD123177 were provided by Dr. R. D. Smith (DuPont-Merck Pharmaceutical Co., Wilmington, DE), and the peptide CGP42112 was a gift from Dr. M. De Gasparo (Ciba-Geigy, Basel, Switzerland). PC12W cells (22) and R3T3 cells (21) were provided by Dr. R. Speth (Washington State University, Pullman, WA) and Dr. D. T. Dudley (Parke Davis, Ann Arbor, MI). Bovine AT1 complementary DNA (cDNA) (4) and AT2 cDNA (6) were provided by Dr. T. Inagami (Vanderbilt University, Nashville, TN).

Cell culture
Bovine adrenal fasciculata cells (BAC) were prepared by sequential treatment of adrenal cortical slices with trypsin (0.15%) (23). The cells were cultured in a chemically defined medium of DMEM/F12 (1:1) supplemented with NaHCO3 (14 mM) and HEPES (10 mM) and containing gentamicin (20 µg/ml), penicillin (100 U/ml), streptomycin (0.1 mg/ml), nystatin (100 U/ml), transferrin (10 µg/ml), insulin (10 µg/ml), and FCS (1%). On the second day of culture, the medium was removed and replaced with serum-free medium. More than 95% of both isolated and cultured cells expressed 3ß-hydroxysteroid dehydrogenase (data not shown), and acute stimulation with either corticotropin or AngII produced about 400 ng/106 cells·2 h cortisol, a specific marker of adrenal fasciculata cells, whereas production of aldosterone, the specific marker of adrenal glomerulosa cells, was less than 2 ng/106 cells·2 h. In both dose-response and time-course experiments, AngII was renewed every 12 h. In experiments in which AngII antagonists were used, they were added 15 min before AngII.

PC12W cells were grown in DMEM/F12 supplemented with antibiotics, 5% FCS, and 10% heat-inactivated horse serum. Cells were plated at a density of 1 x 105 cells/cm2 and cultured for 7–10 days at 37 C under a humidified atmosphere of 5% CO2. Subsequently, cells were cultured in DMEM/F12 supplemented with 2.5% calf serum, 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium, and 1.25 mg/ml BSA. When they approached confluence, PC12W cells were used for radioligand binding assays. R3T3 cells were grown in DMEM/F12 supplemented with antibiotics and 10% FCS. Cells were plated at a density of 1.25 x 105 cells/cm2 and cultured for 5–7 days at 37 C. When they approached confluence, cells were cultured for 3–4 days in DMEM/F12 supplemented with 0.1% BSA.

Radioligand binding assay
AngII was radiolabeled by the Iodogen method and then purified on a diethylaminoethyl-trisacryl column (7). The specific activity of the isolated monoiodinated AngII, as measured by self-displacement in a RRA, was 600–900 Ci/mmol. CGP42112 was radiolabeled by the Iodogen method and then purified by HPLC using a C18 µBondapak column (Millipore, Guyancourt, France) eluted with an acetonitrile gradient of 10–60% in trifluoroacetic acid. The specific activity of the isolated monoiodinated peptide was 800-1000 Ci/mmol.

The receptor assay was performed using 12-well plates containing approximately 0.5 x 106 cells/well. When cells were preincubated with unlabeled ligands, they were washed once with 1 ml 150 mM ice-cold NaCl containing 1% BSA (NaCl/BSA), ice-cold acidic glycine buffer (50 mM glycine and 150 mM NaCl, pH 3.0) for 3 min at 4 C and twice with NaCl/BSA to remove the membrane-bound ligand (13). Binding was carried out for 2 h at room temperature (equilibrium conditions) in 0.5 ml binding medium (DMEM/F12, 0.5% BSA, 0.1% bacitracin, and 10 mM HEPES, pH 7.4) containing approximately 105 cpm labeled ligand. AT1-binding sites were measured using [125I]AngII in the presence of 5 x 10-8 M CGP42112, a concentration at which AT2 receptors were saturated. AT2-binding sites were measured using [125I]CGP42112. Nonspecific binding was evaluated in the presence of 10-6 M AngII or 10-7 M CGP42112. At the end of the incubation, the medium was removed, and the cells were washed three times with NaCl/BSA and then dissolved in 0.5 M NaOH-0.4% sodium deoxycholate. The radioactivity of the binding assays was measured in a {gamma}-counter.

RNA isolation and Northern blot analysis
Total RNA was isolated from cells by the method of Chomczynski and Sacchi (24). For Northern blot analysis, total cytoplasmic RNA (15–40 µg) was subjected to electrophoresis through 1% agarose gels containing 8% formaldehyde. RNA was then transferred to a Hybond-N membrane (Amersham, Les Ulis, France). Membranes with bound RNA were irradiated for 2 min by UV light and baked at 80 C to cross-link the RNA to the filters. Then, the filters were prehybridized for at least 2 h at 42 C in 50% formamide, 5 x SCC (1 x SCC = 0.15 M NaCl and 0.015 M sodium citrate), 5 x Denhart’s solution, 0.5% SDS, 1% glycine, 50 mM NaPO4, and 0.25 mg/ml boiled salmon sperm DNA. Hybridization was carried out at 42 C overnight in hybridization buffer [50% formamide, 20 mM NaPO4 (pH 6.5), 5 x SCC, 1 x Denhart’s solution, 0.5% SDS, 10% dextran sulfate, and 0.1 mg/ml salmon sperm DNA], using either AT1 cDNA (4) or AT2 cDNA (6) as probe. Labeling of these probes in the presence of [{alpha}-32P]deoxy-CTP was performed with a multiprime DNA labeling system (Amersham, Arlington Heights, IL). The blots were washed twice in 2 x SCC buffer containing 0.1% SDS at room temperature for 15 min, once in 0.2 x SCC containing 0.1% SDS at room temperature for 15 min, and finally in 0.2 x SCC containing 0.1% SDS at 42 C for 5–10 min. The blots were exposed to Hyperfilm MP (Amersham) and analyzed by scanning densitometry using a preference HIT (Sebia, Paris, France). Equal loading of RNA samples was confirmed by photographing the ethidium bromide-stained membranes with Polaroid type 55 film (Polaroid, St. Albans, Hertfordshire, UK) and scanning the 28S RNA images on the photograph negatives.

RNA stability transcription rate
To measure the half-life of both AT1 and AT2 mRNAs, cells on the fourth day of culture were incubated for 8 h without or with AngII (10-7 M). The medium was removed and replaced by fresh medium without or with AngII and containing actinomycin D (10 µg/ml) or {alpha}-amanitin (40 µg/ml). At these concentrations, the incorporation of [3H]uridine into trichloroacetic acid-precipitable material after 2-h incubation was 3% and 16%, respectively. At the indicated times, AT1 and AT2 mRNAs were measured by Northern blot.

The RNA stability as well as the rate of transcription were also determined by the method of Johnson et al. (25) with slight modifications. In brief, cells on the fourth day of culture were incubated for 8 h in the absence or presence of AngII. Then, the medium was removed and replaced by fresh medium without or with AngII and containing 4-thiouridine (50 µM, final concentration) and [3H]uridine (3 µCi/µl, final concentration). After 2 h, the medium was aspirated, the cells were washed three times with cold medium, and the RNA was extracted immediately by the method of Chomczynski and Sacchi (24). The amount of total RNA was quantitated by absorbance at 260 nm, and the specific activity (disintegrations per min/µg RNA) was calculated by counting an aliquot. One aliquot of the total RNA (~40 µg) was removed for Northern blot, and the remainder was used for affinity chromatography with mercurated agarose (Affi-Gel 501, Bio-Rad Laboratories, Richmond, CA) to purify newly synthesized RNA. The amount of this newly synthesized RNA as well as its specific activity were measured. For total unfractionated RNA, the same amounts (micrograms) were used for electrophoresis, whereas for the newly synthesized RNA, the same amount of radioactivity was used. The membranes containing both total and newly synthesized RNA were hybridized successively with AT2 and AT1 32P-labeled cDNA. The autoradiographs were quantitated by densitometry scanning. The rate of transcription and the half-life were calculated exactly as described by Johnson et al. (25).

The transcription rate was determined by the ratio of the optical densities of newly synthesized RNA in control and treated cells. The half-life of the specific transcripts was calculated by the following equation: e-kt = [A - (B x C)/A], where A is the optical density of unfractionated RNA, B is the specific activity (optical density per dpm) of newly synthesized RNA, C is the amount (disintegrations per min) of RNA used in A, k is the first order rate constant of degradation (t1/2 of transcript), and t is the time (2 h).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of AngII receptor subtypes and binding sites mRNAs in BAC PC12W
Binding studies using [125I]AngII have shown that BAC contain about 105 sites/cell of a single class of high affinity binding sites (Kd = ~1 nM) (10). However, the results presented in Fig. 1Go show that these cells express both AT1 and AT2 receptor subtypes, because at saturating concentrations, CGP42112, a specific ligand of the AT2 receptor, reduced by 20% the binding of [125I]AngII, whereas DUP753 or losartan, a specific ligand of AT1 receptor, reduced it by 80%. Moreover, in each experimental condition, the remaining binding was completely inhibited by increasing concentrations of the other ligand. BAC also expressed both receptor mRNAs: a single AT1 transcript of 3.3 kilobases (kb) and two AT2 transcripts of 3.8 and 2.3 kb. On the other hand, PC12W expressed only AT2-binding sites and mRNA. Similarly, R3T3 cells, as previously reported (21, 26), expressed only AT2-binding sites and a single transcript of 3.2 kb (data not shown).



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  		Figure 1. AngII receptor subtypes, binding sites, and mRNA in BAC and PC12W cells. Top and middle panels, Displacement of [125I]AngII bound, either in the presence of CGP42115 (10-8 M) by increasing concentrations of DUP753 (r) or in the presence of DUP753 (10-5 M) by increasing concentrations of CGP42112 ({triangleup}). Bottom, Northern blot of increasing concentrations (micrograms) of total RNA from PC12W cells or BAC.

 
Effect of AngII on AT1- and AT2-binding sites and mRNAs in BAC
Treatment of cells with increasing concentrations of AngII caused a dose-dependent inhibition of AT1 and AT2 binding sites and mRNAs (Fig. 2Go). The maximal inhibitory effect was observed at 10-7 M, but the IC50 for AT1- and AT2-binding sites and AT1 mRNA (3 ± 0.4 x 10-9 M) was significantly higher (P < 0.05) than that for AT2 mRNA (2.8 ± 0.3 x 10-10 M). It must be noted that the former IC50 values are similar to the Kd and ID50 for cortisol secretion (27).



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  		Figure 2. AngII dose-response effects on BAC AT1- and AT2-binding sites (top) and mRNAs (middle). Cells were incubated with the indicated concentrations of AngII for 48 h. Then, the cells were incubated with acidic glycine buffer and incubated for 2 h at room temperature with [125I]AngII in the presence of 5 x 10-8 M CGP42112 (AT1-binding sites) or with [125I]CGP42112 (AT2-binding sites). AT1 and AT2 receptor mRNA levels were measured as described in Materials and Methods. The results, expressed as a percentage of control cells, are the mean ± SEM of four to nine different experiments. Bottom panel, Representative Northern blot.

 
However, the most striking differences were observed in the time-course effects of AngII on the four parameters (Fig. 3Go). AT1-binding sites decreased very rapidly, and by 3 h, more than 50% of the surface receptors disappeared. The half-life of these receptors calculated from the experimental points of the first 6 h was about 2 h. Thereafter, a steady state of about 30% of the control remained during the next 18 h. In contrast, the effects of AngII on AT1 mRNA levels appeared after a lag period of about 3 h and were less marked. The calculated half-life after the initial lag period was about 8 h. The effects of AngII on AT2-binding sites were not significant during the first hours of treatment. Thereafter, the surface receptor declined with an apparent half-life of 14–16 h. Similarly, the effects on AT2 mRNA were not significant during the first 6 h of treatment, but then the levels dramatically decreased, with an apparent half-life of 2 h.



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  		Figure 3. Time-course effect of AngII (10-7 M) on BAC AT1- and AT2-binding sites and mRNAs. At the indicated times, the binding sites and mRNA levels were determined as described in Fig. 2Go. The values in the top and middle panels are the mean ± SEM of three to six different experiments. Bottom panel, Representative Northern blot.

 
The inhibitory effects of AngII on both receptor subtypes, binding sites, and mRNAs, were reversible, but the kinetics of recovery were different for AT1 compared to those for AT2 (Fig. 4Go). Thus, most AT1-binding sites and mRNA were recovered within the first 24 h of hormone withdrawal, whereas recovery of AT2-binding sites and mRNA required more than 3 days.



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  		Figure 4. Recovery of BAC AT1- and AT2-binding sites and mRNAs after AngII treatment. Cells were treated for 24 h with 10-7 M AngII. Then, the cells were washed with acidic buffer and refed with fresh medium without hormone. At the indicated times, the numbers of AT1- and AT2-binding sites and mRNA levels were determined. The results are the mean ± SEM of three experiments.

 
Receptor subtype involved in the regulation of AT1 and AT2 receptors by AngII
To investigate which receptor subtype was involved in the effects of AngII on AT1 and AT2 receptors described above, BAC were treated with AngII in the absence or presence of the AT1 antagonist DUP753 or the AT2-specific ligand CGP42112 for 1 day, and at the end of the experimental period, AT1- and AT2-binding sites and mRNAs were measured (Fig. 5Go). Neither DUP753 nor CGP42112 alone had any effect on the four parameters studied. However, DUP753, but not CGP42112, completely blocked the effects of AngII on both receptor subtypes, binding sites, and mRNA. These findings indicate that the AngII-induced down-regulation of both receptor subtypes was exclusively mediated through AT1 receptor. In favor of this hypothesis are the results presented in Table 1Go, showing that in PC12W and R3T3 cells, which express exclusively AT2 receptors, AngII had an effect on AT2-binding sites and mRNA, whereas in BAC, it markedly reduced both parameters.



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  		Figure 5. Regulation of AT1- and AT2-binding sites and mRNAs in BAC by AngII, DUP753, and CGP42112, alone or together. Cells were treated for 24 h with AngII (10-8 M), DUP753 (10-5 M), and CGP42112 (5 x 10-8 M), alone or in combination. At the end of the experiment, the numbers of AT1- and AT2-binding sites and mRNA levels were measured. The results are the mean ± SEM of four to five different experiments. *, P < 0.001 compared to the corresponding control values.

 

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  		Table 1. Effects of AngII on AT2-binding sites and mRNA in BAC, PC12W, and R3T3 cells

 
The lack of effect of AngII on AT2-binding sites in the presence of DUP753 in BAC and of AngII alone in PC12W and R3T3 cells might be due to either a lack of internalization of the occupied receptor or rapid recycling of the internalized receptor. To investigate the first hypothesis, both BAC and PC12W cells were incubated with [125I]AngII (10-9 M) in the absence or presence of DUP753 or CGP42112, and the total membrane-bound (released by acidic washing) and internalized (acid-resistant) hormone levels were determined (Table 2Go). In BAC incubated with [125I]AngII alone, approximately 60% of the hormone was internalized. However, in the presence of CGP42112, which blocks the binding of [125I]AngII to AT2 receptors, the internalized hormone increased to 75% of the total amount bound. In contrast, in the presence of DUP753, which blocks the binding of [125I]AngII to AT1 receptor, no internalization was observed. Similarly, in PC12W cells, the hormone was not internalized regardless of the experimental condition.


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  		Table 2. Internalization of [125I]AngII by BAC and PC12W cells

 
Effect of protein kinase C activation on AngII-binding sites and mRNA in BAC and PC12W cells
In BAC, AngII has been shown to activate the phosphoinositide pathway through the AT1 receptor (27), whereas in PC12W cells, AngII is unable to activate it (28). To elucidate whether activation of protein kinase C (PKC) was involved in the regulation of AT1- and AT2-binding sites and mRNAs, both BAC and PC12W cells were treated for 2 days with either AngII or PMA. The results (Fig. 6Go) show that in both cell types, PMA reduced the number of AT2-binding sites and the level of mRNA. Moreover, in BAC, the effects of AngII and PMA were similar on all parameters studied, except on AT1-binding sites, for which the effects of PMA were less marked than those produced by AngII, probably because PMA was unable to induce the internalization and degradation of AT1 receptors.



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  		Figure 6. Effects of AngII and PMA on AngII-binding sites and mRNAs in BAC and PC12W cells. Cells were treated for 2 days with AngII (10-7 M) or PMA (10-7 M). At the end of the experiment, the numbers of AT1- and AT2-binding sites and mRNA levels were determined. The results are the mean ± SEM of three experiments. a, P < 0.02 compared to control; b, P < 0.02 compared to AngII.

 
Half-life of AT1- and AT2-binding sites
The different kinetics in the decline in the numbers of AT1- and AT2-binding sites in BAC after AngII treatment could be related to a difference in the half-lives of these two receptors. To measure them, BAC were incubated with cycloheximide (10 µg/ml) at a concentration that blocked more than 95% of protein synthesis (data not shown) for 12 h, and at different times AT1- and AT2-binding sites were evaluated. The results (Fig. 7Go) show that the half-lives of AT1 and AT2 were similar (14.8 ± 1.5 and 16.9 ± 2 h, respectively). For comparison, in the same figure are shown the effects of AngII and AngII plus cycloheximide on AT1- and AT2-binding sites. Clearly, the results show that the different effects of the hormone on these two receptors are not related to a difference in their half-lives and that cycloheximide was unable to block the rapid effects of AngII on AT1-binding sites.



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  		Figure 7. Half-lives of AT1- and AT2-binding sites in BAC. Cells were incubated with AngII (10-7 M; X), cycloheximide (10 µg/ml; {square}), or both ({triangleup}). At the indicated times, cells were washed with acidic buffer, and the numbers of AT1- and AT2-binding sites were measured. The results, expressed as a percentage of those in nontreated cells, are the mean ± SD of two independent experiments, each performed in triplicate.

 
Effects of AngII on AT1 and AT2 mRNA stability and gene transcription
The inhibitory effects of AngII on AT1 and AT2 mRNAs could be due to either an increase in the rate of degradation and/or a decrease in the rate of transcription. To evaluate the half-lives of the two mRNAs, control and AngII-treated cells (8 h) were incubated in the presence of actinomycin D (10 µg/ml), and the levels of both mRNAs were evaluated by Northern blot at several times. In the presence of actinomycin D, the half-life of AT1 mRNA was similar in control and AngII-treated cells (8.1 ± 1.1 and 8.2 ± 1.5 h, respectively; n = 3; Fig. 8Go). The half-life of AT2 mRNA in control cells was about 18 h, which was approximately 8-fold longer than the calculated half-life between 8–12 h after the addition of AngII (Fig. 3Go). However, when the 8-h AngII-treated cells were treated with actinomycin D, there was a small increase in the level of AT2 mRNA within the first 2 h. Thereafter, the level of AT2 mRNA declined with an apparent half-life similar to that in control cells. Thus, it appears as if actinomycin D itself may cause a marked stabilization of the AT2 mRNA. Similar results were obtained when {alpha}-amanitin (50 µg/ml) was used as transcription inhibitor (data not shown).



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  		Figure 8. Half-lives of AT1 and AT2 mRNA in BAC. Cells were incubated for 8 h in the absence ({blacksquare}) or presence ({square}) of AngII (10-7 M). Then (time zero), the medium was removed and replaced by fresh medium containing actinomycin D (10 µg/ml) without ({blacksquare}) or with ({square}) AngII. At the indicated times, AT1 and AT2 mRNA levels were evaluated by Northern blot. The results, expressed as a percentage of the results in nontreated cells, are the mean ± SEM of three experiments.

 
To evaluate the effects of AngII on the rate of transcription of the AT1 and AT2 genes, we used the method described by Johnson et al. (25), which also allows evaluation of the half-life of mRNA (Table 3Go and Fig. 9Go). AngII markedly reduced (by 90%) the transcription of AT1 mRNA. It should be noted that similar results were obtained for AT1 mRNA half-life using this method and actinomycin D (see above). Moreover, further analysis of the data, as proposed by Johnson et al. (25), showed that the assumption of steady state on which the determination of half-life depends was fulfilled. The effects of AngII on AT2 transcription and AT2 mRNA half-life were different, i.e. it decreased the rate of transcription by about 30% and the half-life by more than 80%. However, because in this case steady state conditions were lacking, the rate of transcription was probably underestimated, whereas the half-life might have been overestimated.


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  		Table 3. Relative transcription rates and half-life of AT1 and AT2 transcripts in control and AngII-treated (10 h) cells

 


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  		Figure 9. Hybridization of AT1 (right) and AT2 (left) cDNAs to thiol-labeled unfractionated RNA (1, 2) and newly synthesized RNA (3, 4) prepared from control cells (1, 3) and cells treated for 8 h with AngII (10-7 M). Thiol labeling and RNA isolation were performed as described in Materials and Methods.

 
Effects of protein and mRNA synthesis inhibition on AngII-induced down-regulation of AT1 and AT2 mRNA levels
As the effects of AngII on both receptor subtype mRNA levels were observed after a lag period, we hypothesized that they probably required new protein and/or mRNA synthesis. In the experiments presented in Fig. 10Go, cells were treated for 12 h with cycloheximide or actinomycin D in the absence or presence of AngII. The antibiotics were added 10 min before AngII. Cycloheximide alone had very small and nonsignificant effects on AT1 or AT2 mRNA levels, but significantly reduced (P < 0.02) both AT1- and AT2-binding sites (see also Fig. 3Go). Actinomycin D had no effect on either AT1- or AT2-binding sites, but, as expected (see Fig. 8Go), reduced both AT1 and AT2 mRNA levels (P < 0.02). More important cycloheximide almost completely abolished the negative effects of the hormone on both AT1 and AT2 mRNA levels, but not on AT1-binding sites. Actinomycin D also blocked the effects of AngII on AT2 mRNA levels, but not on AT1-binding sites. Whether this antibiotic blocked the inhibitory effects of AngII on AT1 gene transcription is unknown, because the AT1 mRNA levels were similar in cells treated with AngII, actinomycin D, or AngII plus actinomycin D.



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  		Figure 10. Effects of cycloheximide and actinomycin D on AngII-induced down-regulation of AT1- and AT2-binding sites and mRNA levels. Cells were treated for 12 h with AngII (10-7 M), cycloheximide (10 µg/ml), or actinomycin D (10 µg/ml), alone or together. At the end of the experiment, the numbers of AT1- and AT2-binding sites and mRNA levels were evaluated. Top, Mean ± SEM of four experiments. Bottom, Representative Northern blot. a, P < 0.02 vs. control; b, P < 0.02 vs. AngII alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are that AngII down-regulates both AT1 and AT2 mRNAs and binding sites, but with different kinetics and through different mechanisms, and that these effects are mediated exclusively through AT1 receptor subtype. These conclusions are supported by the observation that in BAC, which express both receptor subtypes, the effects of AngII were blocked by AT1 antagonist, whereas in PC12W and R3T3 cells, which express only AT2 receptor, AngII had no effect on AT2 mRNA levels or binding sites.

Many in vivo and in vitro studies have shown that one of the most potent factors affecting AngII receptor protein, as determined by early ligand binding studies (measuring both AT1 and AT2 receptors), is AngII itself (7, 8, 9, 10, 29). In some of these studies it was demonstrated that rapid sequestration and internalization of the hormone-receptor complex were two of the mechanisms responsible for the down-regulation (8, 9, 10, 11). This hormone-induced internalization of the receptor has been recently confirmed using cells stably transfected with both AT1a and AT1b rat cDNAs (30, 31, 32, 33). In some studies it was shown that internalization of the receptor is independent of agonist-activated transduction and that the molecular determinants for agonist-induced receptor internalization are located in a short domain on the cytoplasmic tail (33). In contrast with these negative effects of AngII on its own receptor observed in most of its target tissue in several species, contradictory results have been reported concerning the regulation of the AngII receptor in rat adrenals. An increase in total AngII receptor has been observed after nephrectomy (34) or potassium loading (35), which decrease plasma AngII levels, but also after sodium depletion in rats (36), which increases plasma AngII levels. Similarly, constant infusion of low doses of AngII also increases the number of adrenal AngII receptors (37), whereas at high concentrations the hormone reduced its receptor in adrenal as well as other target tissues (38). The reasons for these apparently contradictory results are not clear, but may be related to a different regulation of AT1 and AT2 receptors expressed in rat adrenals (2) and/or to the fact that in rats and mice, but not in other species, AT1 receptors are encoded by different genes (39, 40), which have different patterns of expression and regulation (see below).

Using specific probes for AT1- and AT2-binding sites and mRNAs, we have been able to evaluate more precisely the kinetics and mechanisms by which AngII regulates both receptor subtypes in BAC. AngII induced a dose- and time-dependent decrease in AT1-binding sites. At maximal concentrations, the initial apparent half-life of AT1 receptor was about 2 h, which was about 8-fold shorter than that observed in control cells in the presence of cycloheximide. As the AT1 mRNA levels during the first 3 h of AngII treatment were decreased by less than 10%, and the AngII-receptor complex (exclusively AT1 receptor; see below) was internalized, the initial rapid decline in AT1-binding sites is probably due to the internalization-degradation process of occupied AT1 receptor. In addition to this rapid effect, the hormone thereafter caused a decrease in AT1 receptor mRNA levels that was also time and dose dependent and, due to an inhibition of the transcription rate, produced no apparent change in the AT1 mRNA half-life. Similarly, in normal human cultured adrenal fasciculata cells (13) and the human tumor adrenal cell line H295 (41), AngII also reduced AT1 mRNA levels.

Regulation of AT1 mRNA has been studied mainly in the rat tissue, and some of the results are contradictory. Sodium depletion, which increases plasma AngII levels, has been reported to either have no effect on AT1a or AT1b mRNA levels in several tissues, including the adrenal (17), or to increase AT1b mRNA in the brain (42), whereas AT1 receptor antagonist administration to intact rat decreases the levels of both mRNAs in aorta and brain and that of AT1b in adrenal (16, 17). On the other hand, bilateral nephrectomy decreases AT1a mRNA levels in brain, liver, and adrenal, but increases those of AT1b in adrenals (16, 18), whereas infusion of AngII increased total AT1 mRNA in adrenal, but had no effect in other tissues (16). In contrast, in cultured rat glomerular mesangial cells (12) and in cultured vascular smooth muscle cells (14, 15), AngII markedly reduced AT1 mRNA levels. Thus, it appears that in the rat, the two AT1 receptor mRNAs are regulated differently, but no clear-cut conclusion can be drawn.

AngII also down-regulated both AT2-binding sites and mRNA, but with different kinetics and through different mechanisms than those involved in the regulation of the AT1 receptor. In BAC, occupancy of only AT2 by either AngII in the presence of the AT1 antagonist DUP753 or by CGP42112 had no effect on AT2-binding sites and mRNA. Similarly, in PC12W and R3T3 cells, which contain only AT2 receptors (21, 22), AngII was unable to modify AT2-binding sites and mRNA. Moreover, no internalization of [125I]AngII was observed when BAC were incubated with the labeled hormone in the presence of DUP753 or when PC12W cells were incubated with the labeled hormone alone. These results confirm previous studies showing the lack of agonist-induced AT2 internalization (33, 43) and demonstrate that the effects of AngII on AT2 receptor are exclusively mediated through the AT1 receptor. Moreover, the half-life of AT2 receptor was similar in cells treated with cycloheximide and those treated with AngII, suggesting that the hormone had no significant effect on the turnover of AT2 protein. Indeed, our results clearly show that AngII after a lag period of 6–8 h dramatically decreased AT2 mRNA levels, mainly by reducing its half-life from 18 h in control cells to about 2 h in AngII-treated cells. These observations suggest that AngII induced the synthesis of labile destabilizing factor(s), which degrades the AT2 mRNA. In favor of this hypothesis is the fact that both cycloheximide and actinomycin D block the effects of AngII on AT2 mRNA levels. Unfortunately, examination of the rate of decay of AT2 mRNA in control and AngII-treated cells in the presence of two different transcription inhibitors did not reveal a difference in the stability of the message, most likely because the transcription inhibitor blocked transcription of the labile destabilizing factors. Similar unexpected results have been reported recently concerning the LH/hCG receptor in rat granulosa cells (44). The calculated half-life of AT2 mRNA by the [3H]uridine-thiouridine method, showing a reduced half-life in AngII-treated cells, favors the above hypothesis. In addition, AngII appears to decrease the rate of transcription of the AT2 gene, but these small changes in transcription cannot explain the rapid and marked changes in AT2 mRNA levels.

Studies concerning the regulation of AT2-binding sites by AngII in other cell types are sparse, and some of the results are conflicting. In rat granulosa cells (20), which contain only AT2 receptor subtypes (19), AngII reduced the number of AT2-binding sites. However, whether this decrease corresponds to receptor loss or to occupancy by the hormone added to the culture is unknown, as in these studies binding measurements were performed in cells not washed with acidic buffer to remove the bound hormone. In cultured bovine thecal cells, which also express only AT2 receptor, AngII had no effect on these receptors (45). In contrast, in R3T3 fibroblasts that selectively express AT2 receptors at confluence, it has been reported that AngII as well as its peptide (Sar1,Ala8-AngII) and nonpeptide (PD123319) antagonists increase AT2 receptor number 2- to 3-fold (21), but decrease by about 25% the levels of AT2 mRNA (26). These surprising results, which are opposite those we observed in BAC, PC12W, and R3T3 cells, are difficult to explain. It must be pointed out that this lack of effect of AngII was observed using different cell passages. Moreover, under the same experimental conditions, both AT2-binding sites and mRNA in PC12W and R3T3 cells were down-regulated by 10% FCS and basic fibroblast growth factor (our unpublished data), as previously shown in rat vascular smooth muscle cells (46) and R3T3 cells (21, 26).

In all cells studied, AT1 receptors are coupled to the phosphoinositide pathway and, therefore, to PKC activation (2, 3, 4), whereas the intracellular effectors of AT2 are still not well defined (2, 5, 6, 27, 43). Our results show that activation of PKC by PMA mimicked most of the effects of AngII in BAC (except on AT1-binding sites) and reduced both the number of AT2-binding sites and mRNA levels in PC12W cells. These findings indicate that AngII regulates its own receptors mainly through PKC. Moreover, the effects of AngII on AT1 and AT2 mRNA required new mRNA and protein synthesis, as they were blocked by both actinomycin D and cycloheximide. In contrast, the rapid effects of AngII on AT1-binding sites were not blocked by any of these antibiotics.

In conclusion, the present findings identify two components of AngII-induced down-regulation of AT1 receptors: rapid internalization-degradation of the occupied receptors and reduction of AT1 mRNA levels due to an inhibition in the rate of transcription. Both of these effects require occupancy of the AT1 receptor. They also show a unique cross-regulation of the AT2 receptor in which AngII, exclusively through the AT1 receptor, caused a decrease in AT2 receptor mainly by decreasing AT2 mRNA stability.


   Acknowledgments
 
The authors thank Drs. R. D. Smith and C. Sweit for providing Losartan, Dr. M. De Gasparo for providing CGP42112, and Dr. T. Inagami for providing AT2 cDNA. We also thank J. Bois for her secretarial help, as well as Dr. J. Carew for editorial assistance.


   Footnotes
 
1 This work was supported by an Institut National de la Santé et de la Recherche Médicale-Merck, Sharpe, and Dohme grant. Back

Received August 16, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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