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(Hypertension. 1998;32:527-533.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Regulation of Angiotensin II Receptor Expression by Nitric Oxide in Rat Adrenal Gland

Makoto Usui; Toshihiro Ichiki; Makoto Katoh; Kensuke Egashira; ; Akira Takeshita

From the Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University Faculty of Medicine, Fukuoka, Japan.

Correspondence to Toshihiro Ichiki, MD, PhD, Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University Faculty of Medicine, 3–1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

	Abstract

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Abstract—We recently reported that administration of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) production, activates the vascular and cardiac renin-angiotensin systems and causes vascular thickening and myocardial hypertrophy in rats with perivascular and myocardial fibrosis. It has been reported that aldosterone may contribute to the development of cardiac fibrosis, but it is not known whether inhibition of NO synthesis affects angiotensin II (Ang II) receptor gene expression and aldosterone secretion. The aim of this study was to investigate the effect of NO inhibition on the expression of Ang II receptors in the adrenal gland and on aldosterone secretion in rats. Wistar King A rats received normal water, L-NAME alone (1 mg/mL in the drinking water), or L-NAME and the 1-adrenergic receptor blocker bunazosin (0.1 mg/mL in the drinking water) for 1 week. After 1 week of treatment with L-NAME, systolic blood pressure, plasma aldosterone concentration (PAC), and mRNA level and number of Ang II type 1 receptor (AT₁-R) were increased. Plasma renin activity, serum angiotensin-converting enzyme activity, and the number of AT₂-R were unchanged. Although addition of bunazosin to L-NAME restored systolic blood pressure to the control level, PAC and AT₁-R numbers remained significantly higher than those of control level. These results suggest that the increased AT₁-R number and PAC induced by the inhibition of NO synthesis were independent of blood pressure and systemic renin-angiotensin system. Therefore, hypertension and myocardial fibrosis induced by NO blockade may be due in part to an elevation of PAC caused by increased AT₁-R in the adrenal gland.

Key Words: nitric oxide • receptors, angiotensin • plasma aldosterone concentration • L-NAME • adrenal gland

	Introduction

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Angiotensin II (Ang II) has a strong vasopressor effect and also regulates electrolyte balance and drinking behavior. Its actions are mediated by specific receptors located on various target organs, including the adrenal gland, kidney, uterus, brain, and arterioles.^{1 2} Recently, it was discovered that Ang II receptors are heterogeneous and could be classified into at least 2 subtypes: Ang II type 1 receptor (AT₁-R) and type 2 receptor (AT₂-R). It is generally accepted that most of the biological effects of Ang II known to date are mediated by the AT₁-R. AT₁-R antagonists reduce blood pressure in renal hypertensive rats and inhibit Ang II–induced aldosterone release from adrenal cortex, epinephrine secretion, and water drinking.^{3 4 5 6 7} Recent molecular cloning of the AT₁-R in rats revealed the existence of 2 subtypes, ie, AT_1A-R and AT_1B-R.^{8 9 10 11 12} The AT₂-R is abundantly expressed in adrenal medulla, but its function has not been identified.

Endothelium plays an important role in the homeostasis of vascular tone by producing endothelium-derived substances.^{13 14 15} An important endothelium-derived relaxing factor has been identified to be nitric oxide (NO) or a related compound.^{16 17} In addition, NO inhibits platelet aggregation and leukocyte adhesion, which suggests that NO may have a negative effect on the growth and/or proliferation of blood vessels.^{13 15 18 19 20 21 22} Several reports have shown that NO antagonizes the biological function of Ang II. NO inhibits Ang II–induced migration of vascular smooth muscle cells.²³ NO also prevents Ang II–induced [³H]thymidine incorporation and cell proliferation in rat mesenteric arteriolar smooth muscle cells.²⁴

We^{25 26 27} and other investigators^{26 27 28 29 30 31} have recently reported that long-term blockade of NO synthesis with chronic administration of N-nitro-L-arginine methyl ester (L-NAME) caused systemic arterial hypertension, microvascular structural changes (medial thickening and perivascular fibrosis), and myocardial hypertrophy in rats and pigs. L-NAME–induced microvascular structural changes were prevented by angiotensin-converting enzyme (ACE) inhibition and Ang II receptor antagonism,^{32 33 34} suggesting that the renin-angiotensin system (RAS) is activated by inhibition of NO. However, it has not been determined whether inhibition of NO synthesis modulates Ang II receptor expression and aldosterone secretion in the adrenal gland. In the present study, we investigated the regulation of adrenal Ang II receptor expression and plasma aldosterone concentration (PAC) in rats treated with L-NAME.

	Methods

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The following protocols were reviewed and approved by the Committee of the Ethics on Animal Experiment in the Faculty of Medicine, Kyushu University, and met the requirements of the Law (No. 105) and Notification (No. 6) of the Government.

Drugs and Materials
We used L-NAME (Sigma Chemical Co), bunazosin (Esai Pharmaceutical Co), CV11974 (Takeda Pharmaceutical Co), PD123319 (Warner-Lambert Co), and [³²P]-dCTP and [¹²⁵I]-Sar,¹Ile⁸-Ang II (New England Nuclear).

Animal Preparation and Tissue Preparation
Eight-week-old male Wistar King A (WKA) rats were obtained from an established colony at the Animal Research Institute of Kyushu University Faculty of Medicine. Three groups of rats were studied; the first group (C group, n=10) received normal drinking water, the second (L group, n=10) received L-NAME in drinking water (1 g/L), and the third (L+B group, n=10) received L-NAME and the 1-adrenergic blocker bunazosin (0.1 g/L) in the drinking water. The daily intake of L-NAME was approximately 100 mg · kg^-1 · d^-1 in the L group. All rats were fed with normal chow and were housed in a viral-antigen–free facility for 1 week. All hemodynamic and biochemical studies were performed after 1 week of treatment.

After 1 week of treatment, systolic blood pressure (SBP; tail-cuff method) and body weight were measured. All rats were anesthetized with an injection of sodium pentobarbital (50 mg/kg IP) and killed by exsanguination. Blood was collected into prechilled tubes containing EDTA disodium salt for the measurements of plasma renin activity (PRA) and plasma aldosterone concentration (PAC). Blood was collected in plain tubes for the measurement of serum ACE activity, serum corticosterone concentration (SCC), and serum potassium and sodium concentrations. The plasma and serum were separated by centrifugation at 3000 rpm for 20 minutes at 4°C and stored at -80°C until measurement of PRA, PAC, serum ACE activity, SCC, and serum potassium and sodium concentrations. PRA was measured as the rate of angiotensin I generation from angiotensinogen, determined with a radioimmunoassay (SRL Co Ltd). PAC was also determined with a radioimmunoassay method (SRL Co Ltd). Serum ACE activities were measured using a fluorometric assay described by Cheung and Cushman³⁵ and Hayashi et al.³⁶ Serum ACE activity was calculated as nanomoles His-Leu generated per milliliter of serum per hour. SCC was also determined with a radioimmunoassay method (SRL Co Ltd). Left and right adrenal glands were removed, frozen in liquid nitrogen, and stored at -80°C. Both adrenal glands from 6 rats in each group were pooled and used for RNA extraction for Northern blot analysis, and adrenal glands from another 4 rats were used for the radioligand binding assay.

cDNA Probes for Northern Blot Analysis
The cDNA probes were prepared as described previously.³⁷ To obtain a rat AT_1A-R–specific probe, total RNA from rat kidney was reverse transcribed (Ready-To-Go T-Primed First-Strand kit, Pharmacia Biotech AB), and the resultant cDNA was amplified by polymerase chain reaction (PCR) with the following primers³⁸ : sense primer 5'-TGGCTTACGACCAAAGGACCA-3' and antisense primer 5'-CAAAGGGAGACTGATGAGATTG-3'. The AT_1B-R–specific probe was prepared in the same way, except that total RNA from rat adrenal gland was used. A noncoding fragment (395 bp; +1246 to +1641) was used as a template for making cDNA probes.⁸ PCR was carried out by 25 cycles of denaturation at 95°C for 60 seconds, annealing at 60°C for 60 seconds, and polymerization at 72°C for 60 seconds. The 379-bp products for AT_1A and 395-bp PCR products for AT_1B were subcloned into the pBluescript II KS(+) vector (Stratagene). The specificity of the AT_1A-R and the AT_1B-R probes was confirmed by a lack of cross-hybridization between AT_1A-R and AT_1B-R cDNAs. The cDNA probe for AT₂ was provided by Dr Tadashi Inagami, Vanderbilt University, Nashville, Tenn.

RNA Extraction and Northern Blots
Total RNA was extracted by the guanidine thiocyanate-phenol-chloroform extraction method (Isogen; Wako Pure Chemical Ltd). Total RNA (20 µg) was electrophoresed in a 1% agarose formaldehyde gel and transferred to a nylon membrane (Hybond-N⁺, Amersham Co). After prehybridization for 2 hours at 42°C in a hybridization buffer containing 50% formamide, 5x Denhardt's solution, 5x SSC (1x SSC is made up of 150 mmol/L NaCl and 15 mmol/L Na citrate), 0.5% SDS, and 1 g/L heat-denatured salmon sperm DNA, the membrane was hybridized with [³²P]-labeled probe in the same hybridization buffer for 20 hours at 42°C and washed twice in 2x SSC/1.0% SDS for 30 minutes at 55°C. Blots were exposed to XAR-5 x-ray film (Eastman Kodak) at -70°C. The membrane was stripped by boiling in 0.1% SDS solution for 5 minutes and rehybridized to a [³²P]-labeled probe for GAPDH cDNA to obtain a reference for the amount of applied RNA. Autoradiographic signals were scanned by a densitometer (Mac Scope, Mitani Co Ltd). Relative gene expression was expressed as the ratio of AT_1A-R, AT_1B-R, or AT₂-R mRNA to GAPDH mRNA.

Ang II Receptor Binding Assay
Details of the experimental procedures used to prepare the membrane fraction and the binding assay were essentially the same as those described previously by Takahashi et al.³⁹ In brief, whole adrenal tissue was excised and homogenized in 20 volumes of ice-cold buffer (0.25 mol/L sucrose containing 5 mmol/L Tris-HCl and 1 mmol/L MgCl₂, pH 7.5) in a Polytron PT-10 (Kinematica) with 3 bouts of 10 seconds each at a setting of 7. The homogenate was centrifuged at 500g for 15 minutes at 4°C. The supernatant was filtered through a double layer of cheesecloth and centrifuged at 50 000g for 30 minutes at 4°C. The resulting pellet was washed twice with an ice-cold incubation buffer (50 mmol/L Tris-HCl, 10 mmol/L MgCl_2, pH 7.5) by repeated resuspension and recentrifugation. The final pellet was resuspended in an ice-cold incubation buffer that contained 2 g/L BSA and 0.2 g/L bacitracin. The membrane preparations were stored at -80°C until use. Binding of [¹²⁵I]-Sar,¹Ile⁸-Ang II to membrane fractions was carried out as follows. The incubation mixture contained 200 µL of membrane preparation (150 to 300 µg of protein), 50 µL of solution with [¹²⁵I]-Sar,¹Ile⁸-Ang II at various concentrations (specific activity, 2200 Ci/mmol), and 50 µL of incubation buffer (for total binding), unlabeled Sar,¹Ile⁸-Ang II (1 µmol/L; for nonspecific binding), PD123319 (10 µmol/L; for AT₁-R binding), or CV11974 (10 µmol/L; for AT₂-R binding). The incubation, started by the addition of the membrane fraction, was carried out for 60 minutes at 25°C and terminated by addition of 2 mL of an ice-cold incubation buffer. The mixtures were subjected to rapid filtration under reduced pressure through glass-fiber Whatman GF/B filters (presoaked in the incubation buffer containing 2 g/L BSA) using a Brandel 24R cell harvester. The filters were immediately washed 4 times with 3 mL of ice-cold incubation buffer. After the filters were dried, the radioactivity trapped on the filters was quantified with an automatic gamma counter (Aloka) at an efficiency of 83%. Specific binding of [¹²⁵I]-Sar,¹Ile⁸-Ang II was defined in terms of total radioactivity minus radioactivity due to nonspecific binding. Each binding assay was carried out in duplicate. The protein concentration was determined by the BCA protein assay (Pierce Chemical Co) using BSA as a standard. To calculate the maximal number of Ang II binding sites (B_max) and the binding constant (K_d), the values of specific binding of Ang II to membrane were plotted according to the method of Scatchard.³⁹

Statistical Analysis
Results are expressed as mean±SEM. Statistical analysis was performed using 1-way ANOVA followed by Fisher's test for multiple comparison. A value of P<0.05 was considered statistically significant.

	Results

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Body Weight and Hemodynamic Variables
Body weights, SBPs, and heart rates before treatment were comparable among the 3 groups (Table 1). Body weight in the C group increased, whereas it did not increase during the 1-week treatment period in the L and L+B group.

View this table: [in this window] [in a new window]   Table 1. Body Weight and Hemodynamic Variables

SBP in the L group increased, but drug treatment did not change SBP in the C and L+B groups. After treatment, heart rate in the L group was less than in the C group but was comparable to that in the L+B group.

AT_1A, AT_1B, and AT₂ mRNA Levels in Adrenal Gland
The expression levels of AT_1A-R, AT_1B-R, and AT₂-R mRNA in the rat adrenal glands were determined by Northern blot analysis (Figures 1 through 3). Densitometric analysis indicated that the ratio of AT_1A-R mRNA to GAPDH mRNA was elevated in the L (1.68±0.15-fold, P<0.01) and L+B (1.58±0.14-fold, P<0.01) groups compared with that in the C group. Similarly, the ratio of AT_1B-R mRNA to GAPDH mRNA was elevated in the L (1.79±0.28-fold, P<0.01) and L+B (1.63±0.20-fold, P<0.05) groups compared with that in the C group. The ratio of AT₂-R mRNA to GAPDH mRNA did not differ among the 3 groups.

View larger version (27K): [in this window] [in a new window]   Figure 1. Northern blot analysis of AT1A-R mRNA in rat adrenal gland. Left, Adrenal AT1A mRNA and GAPDH mRNA in control rats (C), rats treated with L-NAME (L), and rats treated with L-NAME plus bunazosin (L+B). A representative autoradiogram is shown. Right, Densitometric data in which AT1A mRNA levels were normalized by GAPDH mRNA level in 3 groups (n=6). mRNA level of control rats was expressed as 1.0. **P<0.01 vs C group. Values are expressed as mean±SEM. AT1A mRNA was significantly increased in the L group (1.7-fold, P<0.01 vs control) and L+B group (1.6-fold, P<0.01 vs control).

View larger version (26K): [in this window] [in a new window]   Figure 2. Northern blot analysis of AT1B-R mRNA in rat adrenal gland. Left, Adrenal AT1B mRNA and GAPDH mRNA in control rats (C), rats treated with L-NAME (L), and rats treated with L-NAME plus bunazosin (L+B). A representative autoradiogram is shown. Right, Densitometric data in which AT1B mRNA levels were normalized by GAPDH mRNA level in 3 groups (n=6). mRNA level of control rats was expressed as 1.0. *P<0.05, **P<0.01 vs C group. Values are expressed as mean±SEM. AT1B mRNA was significantly increased in the L group (1.8-fold, P<0.01 vs control) and L+B group (L+BUNA; 1.6-fold, P<0.05 vs control).

View larger version (24K): [in this window] [in a new window]   Figure 3. Northern blot analysis of AT2-R mRNA in rat adrenal gland. Left, Adrenal AT2 mRNA and GAPDH mRNA in control rats (C), rats treated with L-NAME (L), and rats treated with L-NAME plus bunazosin (L+B; L+BUNA). A representative autoradiogram is shown. Right, Densitometric data in which AT2 mRNA levels were normalized by GAPDH mRNA level in 3 groups (n=6). AT2 mRNA level was not changed among the 3 groups. mRNA level of control rats was expressed as 1.0.

Ligand Binding
Figure 4 shows the saturation curves and the scatchard plots of the binding of [¹²⁵I]-Sar,¹Ile⁸-Ang II to Ang II receptors in the membrane fraction of adrenal gland from the 3 experimental groups. The calculated maximal binding sites (B_max) and dissociation constants (K_d) are summarized in Table 2. The differences in binding constant were not statistically significant among the 3 groups (Table 2). Numbers of total receptor and AT₁-R in the L and L+B groups were significantly increased. The AT₂-R number was slightly increased in the L and L+B groups; however, this difference was not statistically significant (Table 2 and Figure 4). Therefore, the increased AT₁-R number accounted for the increased Ang II receptor number in the L and L+B groups.

View larger version (21K): [in this window] [in a new window]   Figure 4. Saturation curves (left) and scatchard plot analyses (right) of [125I]-Sar,1Ile8-Ang II binding to Ang II receptors in membrane from rat adrenal glands from the control, L-NAME, and L-NAME plus bunazosin (L+B) groups (n=4). Membrane protein (20 µg) was incubated with [125I]-Sar,1Ile8-Ang II (0.5 to 20 nmol/L) in a final volume of 300 µL assay buffer containing 0.1% BSA. Each point represents mean value of 4 rats in each group. Kd value was not changed among the 3 groups, whereas maximal binding of Ang II (Bmax) was significantly increased in the L (P<0.01 vs control) and L+B (P<0.05 vs control) groups. Data are shown in Table 3.

View this table: [in this window] [in a new window]   Table 2. Characterization of Ang II Receptors in L-NAME–Treated Rats

Figure 5 shows the total, AT₁, and AT₂ receptor density in the adrenal gland of 3 groups measured using 5 nmol/L [¹²⁵I]-Sar,¹Ile⁸-Ang II. Consistent with the changes in total receptor density, the AT₁-R density in the L and L+B groups was significantly (P<0.01) increased compared with that in the C group.

View larger version (29K): [in this window] [in a new window]   Figure 5. Total Ang II, AT1, and AT2 receptor density in rats from the control, L-NAME, and L-NAME plus bunazosin (L+B) groups (n=4). Specific binding to AT1 was measured in the presence of 10 µmol/L PD123319, and specific binding to AT2 was measured in the same way as AT1 in the presence of 10 µmol/L CV11974. *P<0.05, **P<0.01 vs C group. Values are expressed as mean±SEM. AT1 Bmax was significantly increased in the L group (P<0.01 vs control) and L+B group (P<0.01 vs control), but AT2 Bmax was not changed among the 3 groups.

PAC, PRA, Serum ACE Activity, Serum Potassium Concentration, and SCC
PAC was significantly increased in the L and L+B groups compared with that in the C group, but PRA, serum ACE activities, and serum potassium concentration were not different among the 3 groups (Table 3). SCC was slightly increased in the L and L+B groups, but there was no significant statistical difference among the 3 groups (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effects of Treatments on Biochemical Variables

	Discussion

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The present study demonstrates three major findings. First, inhibition of NO synthesis by L-NAME in rats increased both AT_1A-R and AT_1B-R mRNA expression and the AT₁-R number in the adrenal gland, but it did not increase AT₂-R mRNA expression or AT₂-R number. Second, inhibition of NO synthesis increased PAC without significant increases of PRA, serum ACE activity, SCC, or serum potassium concentration. Third, normalization of blood pressure in L-NAME–treated rats did not affect the expression levels of AT_1A-R and AT_1B-R mRNA, the AT₁-R number of the adrenal gland, or PAC. This study is the first to show that inhibition of NO synthesis upregulates the AT₁ receptors in vivo.

As shown previously,³⁴ SBP in the L group was increased after 1 week of treatment (204±9 mm Hg) compared with that in the C group (140±5 mm Hg). The mRNA levels of AT_1A-R and AT_1B-R in the L group were increased 1.7-fold and 1.8-fold, respectively. Although bunazosin reduced blood pressure to a level comparable to that in the C group, it did not affect the mRNA level and the number of AT₁-R. These results suggest that the upregulation of AT₁-R (both mRNA level and receptor number) by L-NAME was independent of blood pressure elevation. Although bunazosin reduced blood pressure to the control level, heart rate in the L+B groups remained decreased. Although the mechanism is not clear, it may suggest that bradycardia induced by L-NAME is not due to an elevation of blood pressure but to inhibition of NO itself.

Our results are consistent with those of Cahill et al.⁴⁰ They reported that NO-generating drugs decreased Ang II receptors in cultured rat vascular smooth muscle cells. However, the precise mechanism of the downregulation of AT₁-R by NO donor or the upregulation of AT₁-R by L-NAME is unknown.

Differential regulation of the transcription of AT_1A-R and AT_1B-R gene has been reported.^{37 41} The AT_1A-R mRNA levels in the kidney are significantly increased, whereas renal AT_1B-R mRNA levels are markedly decreased by low dietary sodium intake.³⁷ The AT_1B-R mRNA levels in the adrenal gland are reduced by treatment with the AT₁-specific antagonist TCV 116, but AT_1A-R mRNA levels are unchanged. In this study, both AT_1A-R and AT_1B-R mRNA levels were upregulated by L-NAME treatment. Therefore, some stimuli upregulate the expression of both receptors, whereas others differentially affect AT_1A-R and AT_1B-R gene transcription. The gene expression of AT₂-R is modulated by many cytokines,⁴² growth factors,⁴³ and protein kinase C.⁴⁴ In the adrenal gland, blockade of NO synthesis did not affect the mRNA level and the receptor number of AT₂-R.

Ang II is a strong secretagogue of aldosterone from adrenal cortex and also important for basal aldosterone release.²⁰ Takemoto et al³⁴ reported that the inhibition of NO synthesis by L-NAME for 8 weeks increased PRA and serum ACE. In this study, we showed that L-NAME administration for 1 week increased PAC but not PRA or serum ACE activity. Bunazosin did not affect PAC. These findings suggest that increased PAC induced by L-NAME was not dependent on the systemic RAS or blood pressure. Because not only Ang II but also corticotropin or serum potassium concentration affect aldosterone secretion, we examined SCC and serum potassium concentration in this study. SCC was slightly increased in the L and L+B groups, but there was no significant statistical difference among 3 groups, and there was no correlation between PAC and SCC. Therefore, it suggests that corticotropin level is not significantly different among 3 groups. In this study, PAC was increased in L and L+B groups, but serum potassium was not increased in these groups. Some compensatory mechanism may work, but the mechanism is not clear. Because PRA, serum ACE activity, and SCC did not change, it is likely that increased PAC caused by L-NAME treatment resulted from the increase in the AT₁-R in the adrenal glands. However, we did not exclude the possibility that the local RAS in the adrenal gland was activated by the blockade of NO synthesis. Our results differed from those of Simmons and Freeman,⁴⁵ which suggested that the aldosterone secretion rate was attenuated in rats treated with L-NAME or N-nitro-L-arginine (L-NNA) compared with that in control animals; however, Simmons and Freeman examined aldosterone secretion rate only 30 minutes after L-NAME or L-NNA injection. We administered L-NAME for 1 week and examined PAC. The difference between our results and those of Simmons and Freeman may be derived from the difference in the period of L-NAME treatment.

Recently, many studies have reported that aldosterone is involved in cardiac hypertrophy and fibrosis, which, together with myocardial cell death, may contribute to progressive myocardial remodeling.^{46 47 48 49 50 51} We have previously shown that chronic treatment with L-NAME in rats caused vascular thickening and myocardial hypertrophy with perivascular fibrosis.³⁴ Therefore, the results of this study suggest the possibility that aldosterone may play a role in perivascular and myocardial fibrosis in these models. However, it is necessary to confirm whether aldosterone antagonist is effective in preventing or attenuating the myocardial remodeling in L-NAME–treated animals. Aldosterone stimulates sodium uptake in the distal tubule of the kidney, resulting in water retention. Therefore, increased PAC may play a role in maintaining high blood pressure in L-NAME–treated rats. Under normal conditions, NO may inhibit aldosterone secretion by suppressing AT₁-R expression.

Our finding that NO modulates the Ang II receptor may have an important implication for the understanding of the role of these molecules in the progression of atherosclerosis. In the rodent model, ACE inhibitor prevents neointimal formation induced by balloon injury. These data suggest that Ang II plays an important role in the growth and proliferation of vascular smooth muscle cells and remodeling of the vascular wall. Our data clearly show that blockade of NO enhanced the expression of Ang II receptor and its biological function in the adrenal gland independent of systemic blood pressure or the systemic RAS. Therefore, it is possible that vascular thickening and perivascular fibrosis observed in rats with chronic treatment of L-NAME are due to upregulation of Ang II receptor in vascular wall. Studies examining the Ang II receptor in vascular wall are in progress and will address the role of Ang II receptor in the pathogenesis of structural changes of coronary artery in L-NAME–treated rats.

In conclusion, these data demonstrate that the inhibition of NO synthesis by L-NAME increased both AT_1A-R and AT_1B-R mRNA expression and AT₁-R number in the rat adrenal gland independent of blood pressure and the systemic RAS. AT₂-R mRNA expression and AT₂-R number did not change with L-NAME, and the inhibition of NO synthesis increased PAC but not PRA or serum ACE activity. Our findings suggest that inhibition of NO synthesis causes AT₁-R upregulation in the adrenal gland and increases PAC. Therefore, L-NAME–induced hypertension and myocardial fibrosis may be partially due to the elevation of PAC induced by increased AT₁-R in the adrenal gland.

	Acknowledgments

 
This study was supported in part by grants-in aid for scientific research (06670725, 06404034, 07557346) from the Ministry of Education, Science, and Culture, Tokyo; by research grants from the Uehara Memorial Foundation, Tokyo, the Study Group of Molecular Cardiology, Tokyo, and the Kaibara Morikazu Science Promotion Foundation, Fukuoka; and a Kimura Memorial Heart Foundation research grant for 1996, Kurume, Japan. The authors would like to thank Fumiko Amano and Tomoko Takebe for their excellent assistance.

Received March 23, 1998; first decision April 2, 1998; accepted April 13, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Herblin WF, Chiu AT, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PB. Angiotensin II receptor heterogeneity. Am J Hypertens. 1991;4:299S–302S.[Medline] [Order article via Infotrieve]

2. Wong PC, Chiu AT, Duncia JV, Herblin WF, Smith RD, Timmermans PB. Angiotensin II receptor antagonists and receptor subtypes. Trends Endocrinol Metab. 1992;3:211–217.[Medline] [Order article via Infotrieve]

3. Dudley DT, Panek RL, Major TC, Lu GH, Bruns RF, Klinkefus BA, Hodges JC, Weishaar RE. Subclasses of angiotensin II binding sites and their functional significance. Mol Pharmacol. 1990;38:370–377.[Abstract]

4. Wong PC, Hart SD, Zaspel AM, Chiu AT, Ardecky RJ, Smith RD, Timmermans PB. Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753 (AII-1) and PD 123177 (AII-2). J Pharmacol Exp Ther. 1990;255:584–592.[Abstract/Free Full Text]

5. Chang RS, Lotti VJ. Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands. Mol Pharmacol. 1990;37:347–351.[Abstract]

6. Gupta P, Franco-Saenz R, Mulrow PJ. Locally generated angiotensin II in the adrenal gland regulates basal, corticotropin, and potassium-stimulated aldosterone secretion. Hypertension. 1995;25:443–448.[Abstract/Free Full Text]

7. Bernstein KE, Alexander RW. Counterpoint: molecular analysis of the angiotensin II receptor. Endocrinol Rev. 1992;13:381–386.[Abstract/Free Full Text]

8. Iwai N, Inagami T. Identification of two subtypes in the rat type I angiotensin II receptor. FEBS Lett. 1992;298:257–260.[Medline] [Order article via Infotrieve]

9. Iwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, Inagami T. Rat angiotensin II receptor: cDNA sequence and regulation of the gene regulation. Biochem Biophys Res Commun. 1991;177:299–304.[Medline] [Order article via Infotrieve]

10. Kakar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD. Angiotensin II type-I receptor subtype cDNAs: differential tissue expression and hormonal regulation. Biochem Biophys Res Commun. 1992;183:1090–1096.[Medline] [Order article via Infotrieve]

11. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of cDNA encoding the vascular type I angiotensin II receptor. Nature. 1991;351:233–236.[Medline] [Order article via Infotrieve]

12. Sandberg K, Ji H, Clark AJL, Shapira H, Catt KJ. Cloning and expression of novel angiotensin II receptor subtype. J Biol Chem. 1992;267:9455–9458.[Abstract/Free Full Text]

13. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;329:2002–2012.[Free Full Text]

14. Dzau VJ, Gibbons GH. Endothelium and growth factors in vascular remodeling of hypertension. Hypertension. 1991;18(suppl III):III-115–III-121.

15. Lüscher TF, Noll G. Endothelial dysfunction in coronary circulation. J Cardiovasc Pharmacol. 1994;24(suppl 3):S16–S26.

16. Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333:664–666.[Medline] [Order article via Infotrieve]

17. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release account for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526.[Medline] [Order article via Infotrieve]

18. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774–1777.

19. McNamara DB, Bedi B, Aurora H, Tena L, Ignarro LJ, Kadowitz PJ, Akers DJ. L-Arginine inhibits balloon catheter-induced intimal hyperplasia. Biochem Biophys Res Commun. 1993;193:291–296.[Medline] [Order article via Infotrieve]

20. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168–1172.

21. Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753–759.[Abstract/Free Full Text]

22. Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau V. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137–1141.[Abstract/Free Full Text]

23. Dubey RK, Jackson EK, Lüscher TF. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. J Clin Invest. 1995;96:141–149.

24. Dubey RK, Overbeck HW. Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. Cell Tissue Res. 1994;275:133–141.[Medline] [Order article via Infotrieve]

25. Ito A, Egashira K, Kadokami T, Fukumoto Y, Takayanagi T, Nakaike R, Kuga T, Sueishi K, Shimokawa H, Takeshita A. Chronic inhibition of endothelium-derived nitric oxide synthesis causes coronary microvascular structural changes and hyperreactivity to serotonin in pigs. Circulation. 1995;92:2636–2644.[Abstract/Free Full Text]

26. Numaguchi K, Egashira K, Takemoto M, Kadokami T, Shimokawa H, Sueishi K, Takeshita A. Chronic inhibition of nitric oxide synthesis causes pathologic coronary microvascular remodeling and myocardial fibrosis. Hypertension. 1995;26(pt 1):957–962.

27. Kadokami T, Egashira K, Kuwata K, Fukumoto Y, Kozai T, Yasutake H, Kuga T, Shimokawa H, Sueishi K, Takeshita A. Altered serotonin receptor subtypes contribute to microvascular hyperreactivity to serotonin in pigs with chronic inhibition of nitric oxide. Circulation. 1996;94:182–189.[Abstract/Free Full Text]

28. Arnal JF, Warin L, Michel JB. Determinants of aortic cyclic guanosine monophosphate in hypertension induced by chronic inhibition of nitric oxide synthase. J Clin Invest. 1992;90:647–652.

29. Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest. 1992;90:278–281.

30. Bank N, Aynedjian HS, Khan GA. Mechanism of vasoconstriction induced by chronic inhibition of nitric oxide in rats. Hypertension. 1994;24:322–328.[Abstract/Free Full Text]

31. Ribeiro MO, Antunes E, Nicci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension. Hypertension. 1992;20:298–303.[Abstract/Free Full Text]

32. Arnal JF, Amrani AIE, Chatellier G, Menard J, Michel JB. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension. 1993;22:380–387.[Abstract/Free Full Text]

33. Pollock DM, Polakowski JS, Divish BJ, Opgenorth TJ. Angiotensin blockade reverses hypertension during long-term nitric oxide synthase inhibition. Hypertension. 1993;21:660–666.[Abstract/Free Full Text]

34. Takemoto M, Egashira K, Usui M, Numaguchi K, Tomita H, Tsutsui H, Shimokawa H, Sueishi K, Takeshita A. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. J Clin Invest. 1997;99:278–287.[Medline] [Order article via Infotrieve]

35. Cheung HS, Cushman DW. Inhibition of homogenous angiotensin-converting enzyme of rabbit lung by synthetic venom peptides of Bothrops jararaca. Biochem Biophys Acta. 1973;293:451–463.[Medline] [Order article via Infotrieve]

36. Hayashi K, Nunami K, Kato J, Yoneda N, Kubo M, Ochiai T, Ishida R. Studies of angiotensin-converting enzyme inhibitors: synthesis and angiotensin-converting enzyme inhibitory activities of 3-acyl-1-alkyl-2-oxoimidazolidine-4-carboxylic acid derivatives. J Med Chem. 1989;32:289–297.[Medline] [Order article via Infotrieve]

37. Du Y, Yao A, Guo D, Inagami T, Wang DH. Differential regulation of angiotensin II receptor subtypes in rat kidney by low dietary sodium. Hypertension. 1995;25:872–877.[Abstract/Free Full Text]

38. Du Y, Guo DF, Inagami T, Speth RC, Wang DH. Regulation of Ang II-receptor subtype and its gene expression in adrenal gland. Am J Physiol. 1996;271:H440–H446.[Abstract/Free Full Text]

39. Takahashi M, Norota I, Endoh M. Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium. Naunyn Schmiedebergs Arch Pharmacol. 1991;343:669–673.[Medline] [Order article via Infotrieve]

40. Cahill PA, Redmond EM, Foster C, Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol. 1995;288:219–229.[Medline] [Order article via Infotrieve]

41. Kitami Y, Okura T, Marumoto K, Wakamiya R, Hiwada K. Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat. Biochem Biophys Res Commun. 1992;188:446–452.[Medline] [Order article via Infotrieve]

42. Ichiki T, Kambayashi Y, Inagami T. Transcription of the rat angiotensin II type 2 receptor gene. Biochem Biophys Res Commun. 1996;222:566–571.[Medline] [Order article via Infotrieve]

43. Camp HS, Dudley DT. Modulation of angiotensin II receptor (AT₂) mRNA levels in R3T3 cells. Receptor. 1995;5:123–132.[Medline] [Order article via Infotrieve]

44. Kijima K, Matsubara H, Murasawa S, Maruyama K, Ohkubo N, Yasukiyo M, Inada M. Regulation of angiotensin II type 2 receptor gene by the protein kinase C–calcium pathway. Hypertension. 1996;27:529–534.[Abstract/Free Full Text]

45. Simmons JC, Freeman RH. L-Arginine analogues inhibit aldosterone secretion in rats. Am J Physiol. 1995;268:R1137–R1142.[Abstract/Free Full Text]

46. Robert V, Silvestre JS, Charlemagne D, Sabri A, Trouve P, Wassef M, Swynghedauw B, Delcayre C. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension. 1995;26:971–978.[Abstract/Free Full Text]

47. Funder JW. Mineralocorticoid receptors and hypertension. J Steroid Biochem Mol Biol. 1995;53:53–55.[Medline] [Order article via Infotrieve]

48. Nicoletti A, Heudes D, Hinglais N, Appay MD, Philippe M, Sassy PC, Bariety J, Michel JB. Left ventricular fibrosis in renovascular hypertensive rats: effect of losartan and spironolactone. Hypertension. 1995;26:101–111.[Abstract/Free Full Text]

49. Young M, Fullerton M, Dilley R, Funder J. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest. 1994;93:2578–2583.

50. Brilla CG, Matsubara LS, Weber KT. Antifibrotic effects of spironolactone in preventing myocardial fibrosis in systemic arterial hypertension. Am J Cardiol. 1993;71:12A–16A.[Medline] [Order article via Infotrieve]

51. Brilla CG, Weber KT. Reactive and reparative myocardial fibrosis in arterial hypertension in the rat. Cardiovasc Res. 1992;26:671–677.[Abstract/Free Full Text]

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