AJP - Renal Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F121-F127, 2007. First published March 27, 2007; doi:10.1152/ajprenal.00478.2006
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F121    most recent
00478.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, D.
Right arrow Articles by Pandey, K. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, D.
Right arrow Articles by Pandey, K. N.

Guanylyl cyclase/natriuretic peptide receptor-A gene disruption causes increased adrenal angiotensin II and aldosterone levels

Di Zhao, Elangovan Vellaichamy, Naveen K. Somanna, and Kailash N. Pandey

Department of Physiology, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana

Submitted 5 December 2006 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Disruption of the guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) gene leads to elevated arterial blood pressure and congestive heart failure in mice lacking NPRA. This study was aimed at determining whether Npr1 (coding for GC-A/NPRA) gene copy number affects adrenal ANG II and aldosterone (Aldo) levels in a gene-dose-dependent manner in Npr1 gene-targeted mice. Adrenal ANG II and Aldo levels increased in 1-copy mice compared with 2-copy mice, but decreased in 3-copy and 4-copy mice. In contrast, renal ANG II levels decreased in 1-copy (25%), 3-copy (38%), and 4-copy (39%) mice compared with 2-copy mice. The low-salt diet stimulated adrenal ANG II and Aldo levels in 1-copy (20 and 2,441%), 2-copy (15 and 2,339%), 3-copy (20 and 424%), and 4-copy (31 and 486%) mice, respectively. The high-salt diet suppressed adrenal ANG II and Aldo levels in 1-copy (46 and 29%) and 2-copy (38 and 17%) mice. On the other hand, the low-salt diet stimulated renal ANG II levels in 1-copy (45%), 2-copy (45%), 3-copy (59%), and 4-copy (48%) mice. However, the high-salt diet suppressed renal ANG II levels in 1-copy (28%) and 2-copy (27%) mice. In conclusion, NPRA signaling antagonizes adrenal ANG II and Aldo levels in a gene-dose dependent manner. Increased adrenal ANG II and Aldo levels may play an important role in elevated arterial blood pressure and progressive hypertension, leading to renal and vascular injury in Npr1 gene-disrupted mice.

salt diets; gene duplication

ATRIAL NATRIURETIC PEPTIDE (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) constitute the natriuretic peptide family (6, 40, 45). ANP and BNP are mainly released from the heart (7, 27, 46), whereas CNP is produced in endothelial cells (45). Three distinct natriuretic peptide receptors have been identified and characterized by molecular cloning; these are natriuretic peptide receptor-A (NPRA), natriuretic peptide receptor-B (NPRB), and natriuretic peptide receptor-C (NPRC) (12, 38, 41). ANP and BNP bind to NPRA, a membrane-bound form of guanylyl cyclase also known as GC-A (10, 35). CNP binds to NPRB, which is also a membrane-bound form of guanylyl cyclase type of receptor called GC-B (12, 16, 41). All three of the natriuretic peptides also bind to NPRC, which lacks guanylyl cyclase activity (12, 22). ANP binding to NPRA increases intracellular second-messenger cGMP concentration by enhanced guanylyl cyclase activity (21, 33, 34, 42). The major effects of ANP-NPRA signaling are natriuretic, diuretic, vasodilatory, antifibrotic, and antihypertrophic (19, 30, 33, 48). Npr1 is the gene that codes for NPRA (13, 14). It has been reported that Npr1 expression affects arterial blood pressure in a gene-dose-dependent manner in Npr1 gene-targeted mice (30, 31, 44). Our previous studies have indicated that NPRA signaling exerts protective effects with respect to blood pressure and cardiovascular regulation and plays important roles in other physiological functions (30, 37, 43, 48).

The adrenal gland regulates blood volume homeostasis and blood pressure mainly by secreting aldosterone (Aldo). A shift has occurred in recent years from an emphasis on the function of the circulating renin-angiotensin system (RAS) to a focus on the local tissue RAS in the brain, heart, peripheral blood vessels, adrenal glands, and kidney (39). Sodium restriction has been found to increase adrenal renin activity and Aldo production in parallel (8) and to increase angiotensin II (ANG II) secretion from the zona glomerulosa cells (5). Nephrectomy has been shown to increase adrenal renin activity via increased serum potassium but to decrease plasma renin to undetectable levels (8, 9). Although some studies have shown that adrenal ANG II may be important in Aldo production through a paracrine or autocrine effect (5, 24), the function of the adrenal RAS in regulating adrenal function still awaits further study.

ANP exerts its biological activity by binding to NPRA, which is predominantly located in the renal glomeruli and adrenal zona glomerulosa (4). ANP inhibits adrenal Aldo synthesis (1, 3, 11) and renal renin secretion (22). It is likely that the inhibitory effect of ANP on Aldo synthesis depends on the functionally active NPRA (32). Adrenal renin activity and mRNA expression were found to be increased in Npr1 gene-disrupted mice compared with wild-type mice (43). Since ANP-NPRA signaling counteracts the RAS in a tissue-specific manner (44), we investigated adrenal ANG II and Aldo levels in Npr1 gene-disrupted and gene-duplicated mice. We also studied the effect of a low- or high-salt diet on adrenal ANG II and Aldo levels in Npr1 gene-disrupted and gene-duplicated mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Npr1 gene-targeted mice were generated by homologous recombination as previously described (30). All mice were littermate progenies of the C57/BL6 genetic background. Mice were bred in the vivarium at Tulane University Health Sciences Center. We used female mice 24–32 wk old. The following are the mice genotypes used: 0-copy (–/–) is a homozygous mutant allele; 1-copy (+/–) is a heterozygous allele; 2-copy (+/+) is a wild-type allele; 3-copy (++/+) is a gene-duplicated heterozygous allele; and 4-copy (++/++) is a gene-duplicated homozygous allele. Mice were maintained at a 12:12-h alternating light-dark schedule (light from 6 AM to 6 PM) at 25°C. They were given a low-salt (0.05% NaCl), normal-salt (0.3% NaCl), or high-salt (8% NaCl) diet along with tap water during the 3 wk of this study. All protocols were approved by the Institutional Animal Care and Use Committee at Tulane University Health Sciences Center.

Blood pressure measurement. We measured the systolic blood pressures of Npr1 gene-targeted mice every other day during the 3-wk experimental period by the noninvasive computerized tail-cuff method with Visitech 2000 (Apex, NC) as previously described (17, 43). An average systolic blood pressure level of 5 sessions/day was calculated for analysis after 7 days of training for blood pressure measurement.

Adrenal gland, kidney, and plasma collection. Animals under CO2 anesthesia were killed by cardiac puncture. Blood was collected with 10 µl 0.25 M EDTA. After centrifugation at 3,000 rpm for 30 min, plasma was separated and kept at –80°C until used. Adrenal glands and kidneys were removed, weighed, and frozen in liquid nitrogen and kept at –80°C until used.

Adrenal and renal ANG II extraction. Adrenal glands and kidneys were rinsed in 0.5 ml buffer and homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) at a setting of 10 (3 times) for 30 s at 4°C in 1.5 ml buffer containing 10 mM pyrophosphate, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM EDTA (36). The supernatants were collected after centrifugation at 40,000 g for 40 min at 4°C. We determined the protein concentrations of adrenal and renal extract using a protein assay reagent (Bio-Rad, Hercules, CA). We used 200-mg Sep-Pak C18 columns (Waters Associates, Milford, MA) for ANG II extraction; the columns were equilibrated with 3 ml 0.1% trifluoroacetic acid (TFA) and washed with 10 ml 0.1% TFA-1% NaCl. The 1 ml adrenal or renal extract was loaded and eluted with a 2-ml mixture of methanol:water:TFA (80:19:1). Eluates were dried overnight in a Speed-Vac centrifuge. The residues were kept at –20°C and for ANG II assay were dissolved into 250 µl 100 mM Tris-acetate buffer (pH, 7.5) containing 1 mM PMSF, 0.02% sodium azide, and 0.1% bovine serum albumin.

Adrenal Aldo extraction. A 0.5-ml adrenal extract and a 5-ml mixture of hexane:ethyl acetate (3:2) were pipetted into a glass tube and vortexed vigorously for 1 min. The 4-ml organic layer was transferred into another glass tube and dried overnight in a Speed-Vac centrifuge. The residues were kept at –20°C and for the Aldo assay were dissolved into 230-µl Aldo zero standards (Diagnostic System, Webster, TX).

ANG II and Aldo assay. We measured adrenal, renal, and plasma ANG II levels using radioimmunoassay (RIA) as previously reported (36, 43). Briefly, bound and free ANG II peptides were separated by dextran-coated charcoal after a 48 h of incubation at 4°C. The supernatants were counted by a gamma counter for 1 min. The results are presented as femtomoles per milligram of adrenal or renal protein content. Adrenal and plasma Aldo levels were assayed as described before (43) using the RIA Kit (Diagnostic System, Webster, TX) and are presented as picograms Aldo per milligram of protein content.

Statistical analysis. The results are expressed as means ± SE. The differences between groups were determined by ANOVA combined with Dunnett's multiple comparison post hoc test using GraphPad PRISM (version 4.0; GraphPad Software, San Diego, CA). A value of P < 0.05 was used as the criterion to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of different salt diets on systolic blood pressure. With mice receiving a normal-salt diet, systolic blood pressure (mmHg) increased in 1-copy mice (12%, 114.0 ± 1.9, P < 0.01) compared with 2-copy mice (102.1 ± 2.0), but decreased in 3-copy (9%, 92.7 ± 1.8, P < 0.01) and 4-copy mice (16%, 86.2 ± 2.2, P < 0.01) (Fig. 1A). With a low-salt diet, systolic blood pressures increased in 1-copy mice (11%, 116.0 ± 4.4, P < 0.01) compared with 2-copy mice (104.3 ± 2.1), but decreased in 3-copy (10%, 93.7 ± 1.8, P < 0.01) and 4-copy mice (18%, 85.3 ± 2.6, P < 0.001) (Fig. 1B). Similarly, with a high-salt diet, systolic blood pressures increased in 1-copy mice (13%, 126.9 ± 2.3, P < 0.05) compared with 2-copy mice (112.2 ± 3.2), but decreased in 3-copy (16%, 94.7 ± 2.8, P < 0.01) and 4-copy mice (24%, 85.9 ± 2.4, P < 0.001) (Fig. 1C). We compared blood pressures in relation to different salt diets in the same Npr1 gene copy number mice. The high-salt diet increased systolic blood pressures in 1-copy (11%, P < 0.01) and 2-copy mice (10%, P < 0.05) compared with a normal-salt diet (Fig. 1, A and C), whereas the low-salt diet did not alter systolic blood pressures in 1-copy, 2-copy, 3-copy or 4-copy mice (Fig. 1, A and B).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Systolic blood pressures in Npr1 gene-targeted mice fed a normal-salt (A), low-salt (B), or high-salt (C) diet: The mice used were Npr11-copy (+/–): gene-disrupted heterozygous allele; 2-copy (+/+): wild type allele; 3-copy (++/+): gene-duplicated heterozygous allele; and 4-copy (++/++): gene-duplicated homozygous allele. These mice were fed a low-salt (0.05%), normal-salt (0.3%), or high-salt (8%) diet. Bars indicate means ± SE values for the representative genotypes. N, no. of mice. To study the effect of Npr1 gene expression on tissue RAS, 2-copy wild-type mice were used as controls. Comparisons were made between 2-copy and other Npr1 mice genotypes fed a diet with the same salt content: **P < 0.01. ***P < 0.001. To study the effect of a low- or high-salt diet on the tissue renin-angiotensin system (RAS), mice fed a normal-salt were the control group. Comparisons were made among Npr1 mice having the same number of gene copies but fed a normal-, low-, or high-salt diet. {dagger}P < 0.05. {dagger}{dagger}P < 0.01.

 
Adrenal ANG II level with different salt diets. With a normal-salt diet, adrenal ANG II levels (fmol/mg protein) increased in 1-copy mice (15%, 74.8 ± 2.7, P < 0.05) compared with 2-copy mice (65.1 ± 2.7), but decreased in 3-copy (17%, 53.9 ± 2.7, P < 0.05) and 4-copy mice (33%, 43.7 ± 4.7, P < 0.01) (Fig. 2A). Similarly, adrenal ANG II levels increased with a low-salt diet in 1-copy mice (20%, 89.8 ± 4.7, P < 0.05) compared with 2-copy mice (74.8 ± 3.0), but decreased in 3-copy (14%, 64.6 ± 2.2, P < 0.05) and 4-copy mice (24%, 57.2 ± 3.1, P < 0.01) (Fig. 2B). On the other hand, with a high-salt diet there were no statistical differences among adrenal ANG II levels in 1-copy (40.6 ± 3.3), 3-copy (40.9 ± 5.8), or 4-copy mice (39.6 ± 4.7) compared with 2-copy mice (40.1 ± 4.1) (Fig. 2C).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 2. Adrenal ANG II levels in Npr1 gene-targeted mice fed a normal-salt (A), low-salt (B), or high-salt (C) diet. Bars indicate means ± SE values for the representative genotypes. N, no. of mice. Comparisons were made among different Npr1 mice genotypes with same salt diet. *P < 0.05. **P < 0.01. Statistical comparisons were performed among Npr1 mice having the same number of gene copies but fed different salt diets. {dagger}P < 0.05. {dagger}{dagger}P < 0.01. {dagger}{dagger}{dagger}P < 0.001.

 
In Npr1 mice with different numbers of gene copies, the low-salt diet increased adrenal ANG II levels in 1-copy (20%, P < 0.05), 2-copy (15%, P < 0.05), 3-copy (20%, P < 0.05), and 4-copy mice (31%, P < 0.05) compared with levels in those given a normal-salt diet (Fig. 2, A and B). In contrast, the high-salt diet decreased adrenal ANG II levels in 1-copy (46%, P < 0.001) and 2-copy mice (38%, P < 0.01), but not in 3-copy and 4-copy mice (Fig. 2, A and C).

Adrenal Aldo level with different salt diets. With a normal-salt diet, adrenal Aldo levels (pmol/mg protein) were increased in 1-copy mice (38%, 8.7 ± 0.8, P < 0.05) compared with 2-copy mice (6.3 ± 0.2), but decreased in 3-copy (13%, 5.5 ± 0.3, P < 0.05) and 4-copy mice (38%, 3.8 ± 0.4, P < 0.001) (Fig. 3A). Similarly, with a low-salt diet, adrenal Aldo levels were increased in 1-copy mice (44%, 221.4 ± 19.6, P < 0.05) compared with 2-copy mice (153.6 ± 20.9), but decreased in 3-copy (81%, 28.6 ± 4.0, P < 0.001) and 4-copy mice (85%, 22.8 ± 3.7, P < 0.001) (Fig. 3B). However, with a high-salt diet, adrenal Aldo levels in 1-copy (6.2 ± 0.7), 3-copy (4.8 ± 0.4), and 4-copy mice (4.2 ± 0.5) did not statistically differ from levels in 2-copy mice (5.2 ± 0.3) (Fig. 3C).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Adrenal aldosterone levels in Npr1 gene-targeted mice fed a normal salt (A), low-salt (B), or high-salt (C) diet. Bars indicate means ± SE values for the representative genotypes. N, no. of mice. Comparisons were made among different Npr1 mice genotypes fed the same salt diet. *P < 0.05. ***P < 0.001. Comparisons were made among Npr1 mice having the same number of gene copies but fed different salt diets. {dagger}P < 0.05. {dagger}{dagger}P < 0.01. {dagger}{dagger}{dagger}P < 0.001.

 
In Npr1 mice with different numbers of gene copies, the low-salt diet increased adrenal Aldo levels compared with levels in those fed a normal-salt diet (Fig. 3, A and B). Specifically, the low-salt diet increased the Aldo level in 1-copy mice by 2,440.6%, or 24.4-fold (P < 0.001); 2-copy mice by 2,338.9%, or 23.4-fold (P < 0.001); 3-copy mice by 424.0%, or (4.2-fold) (P < 0.001); and 4-copy mice by 485.7%, or 4.9-fold (P < 0.001). In contrast, the high-salt diet decreased adrenal Aldo levels in 1-copy (29.1%, P < 0.05) and 2-copy mice (17.2%, P < 0.05), but not in 3-copy or 4-copy mice (Fig. 3, A and C).

Renal ANG II level with different salt diets. With a normal-salt diet, renal ANG II levels (fmol/mg protein) were decreased in 1-copy (25%, 7.7 ± 0.8, P < 0.05), 3-copy (38%, 6.4 ± 0.8, P < 0.01), and 4-copy mice (39%, 6.3 ± 0.7, P < 0.01) compared with 2-copy (10.3 ± 0.8) mice (Fig. 4A). Similarly, with a low-salt diet, renal ANG II levels were decreased in 1-copy (25%, 11.2 ± 1.0, P < 0.05), 3-copy (32%, 10.1 ± 1.6, P < 0.05), and 4-copy mice (38%, 9.2 ± 1.1, P < 0.01) compared with 2-copy (14.9 ± 1.0) mice (Fig. 4B). With a high-salt diet, although renal ANG II levels were decreased in 1-copy mice (26%, 5.6 ± 0.6, P < 0.01), only minor statistical differences were found in 3-copy (7.0 ± 0.9) and 4-copy (6.3 ± 0.7) mice compared with 2-copy mice (7.5 ± 0.6) (Fig. 4C).


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 4. Renal ANG II levels in Npr1 gene-targeted mice fed a normal-salt (A), low-salt (B), or high-salt (C) diet: Bars indicate means ± SE values for the representative genotypes. N, no. of mice. Comparisons were made among different Npr1 mice genotypes fed the same salt diet. *P < 0.05. **P < 0.01. Statistical comparisons were performed among Npr1 mice having the same number of gene copies but fed different salt diets. {dagger}P < 0.05.

 
In Npr1 mice, the low-salt diet increased renal ANG II levels in 1-copy (45%, P < 0.05), 2-copy (45%, P < 0.05), 3-copy (59%, P < 0.05), and 4-copy mice (48%, P < 0.05) compared with levels when mice were fed a normal-salt diet (Fig. 4, A and B). However, the high-salt diet decreased renal ANG II levels in 1-copy (28%, P < 0.05) and 2-copy mice (27%, P < 0.05), but not in 3-copy or 4-copy mice (Fig. 4, A and C).

Plasma ANG II and Aldo levels with different salt diets. The levels of plasma ANG II and Aldo with different salt diets are shown, respectively, in Tables 1 and 2. With a normal-salt diet, plasma ANG II levels decreased in 1-copy (45%), 3-copy (25%), and 4-copy (32%) mice compared with 2-copy mice (Table 1). In contrast, plasma Aldo levels increased only in 1-copy mice (33%) compared with 2-copy mice, while levels in 3-copy (36%) and 4-copy (48%) mice decreased (Table 2). With a low-salt diet, plasma ANG II levels also decreased in 1-copy (20%), 3-copy (17%), and 4-copy (26%) mice compared with 2-copy mice. Conversely, plasma Aldo levels increased only in 1-copy (45%) mice compared with 2-copy mice, but decreased in 3-copy (25%) and 4-copy (30%) mice. In contrast, with a high-salt diet, plasma ANG II levels decreased in 1-copy (55%) mice compared with 2-copy mice; however, the plasma ANG II levels in 3-copy and 4-copy mice did not statistically differ from that in 2-copy mice. Similarly, plasma Aldo levels in 1-copy, 3-copy, and 4-copy mice did not statistically differ from that in 2-copy mice with a high-salt diet.


View this table:
[in this window]
[in a new window]

  		Table 1. Plasma angiotensin II levels in Npr1 gene-disrupted and gene-duplicated mice fed different salt diets

 

View this table:
[in this window]
[in a new window]

  		Table 2. Plasma aldosterone levels in Npr1 gene-disrupted and gene-duplicated mice fed different salt diets

 
We also analyzed the effect of different salt diets on plasma ANG II and Aldo levels within the same Npr1 gene copy number mice. Compared with the levels obtained with a normal-salt diet, the low-salt diet increased plasma ANG II levels in 1-copy (86%), 2-copy (28%), 3-copy (43%), and 4-copy (41%) mice (Table 1). Aldo levels were also greatly increased in 1-copy (184%), 2-copy (159%), 3-copy (206%), and 4-copy (250%) mice fed with a low-salt diet. However, the high-salt diet decreased plasma ANG II levels in 1-copy (48%) and 2-copy mice (37%). Similarly, Aldo levels were also decreased in 1- copy (54%) and 2-copy mice (42%) kept on a high-salt diet (Table 2).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results demonstrate that adrenal ANG II and Aldo levels are increased in 1-copy mice lacking one Npr1 allele compared with levels in 2-copy mice, but that ANG II and Aldo levels are decreased in gene-duplicated 3-copy and 4-copy mice in a gene-dose dependent manner. In line with these findings, our previous studies have also shown that the adrenal renin activity is increased in Npr1 null mutant mice (43). Previous studies have shown that adrenal RAS greatly influences the production of Aldo both in vitro and in vivo (23, 24). The same studies indicated that antagonism of ANP-NPRA signaling on the adrenal RAS may lead to the inhibition of adrenal Aldo production and secretion. This may explain, at least in part, our finding that compared with levels in wild-type mice, adrenal Aldo levels were increased in Npr1 gene-disrupted mice but decreased in Npr1 gene-duplicated mice.

In the present study, a low-salt diet stimulated adrenal ANG II and Aldo levels in 1-, 2-, 3-, and 4-copy mice compared with the levels in mice given a normal-salt diet. However, a high-salt diet suppressed adrenal ANG II and Aldo levels in 1- and 2-copy mice, but not in 3- and 4-copy mice compared with levels obtained in mice given a normal-salt diet. Indeed, we were surprised by the significant response of adrenal aldosterone levels with the low-salt diet. It is interesting that adrenal aldosterone levels were increased >20-fold in 1- and 2-copy mice given a low-salt diet, but increased only about 4-fold in 3- and 4-copy mice. This suggested that in mice fed the low-salt diet, the elevation of adrenal Aldo level was effectively attenuated by increased Npr1 gene copy number. These results suggested that the Npr1 gene copy number affects adrenal Aldo level, especially with a low-salt diet. Sodium restriction increases adrenal renin activity, ANG II release, and Aldo production (5, 8, 24). In a single-cell study, superfused zona glomerulosa cells from rats kept on a low-sodium diet secreted 1.5-fold higher ANG II than did cells from rats kept on a high-sodium diet (23). The increased adrenal ANG II concentration induced by salt restriction may have led to an increase in Aldo production. On the other hand, compared with a low-sodium diet, a high-sodium diet has been shown to decrease the percentage of secretory zona glomerulosa cells (23).

Adrenal Aldo levels were also affected by Npr1 gene copy number in mice given the high-salt diet. Specifically, adrenal Aldo levels in 1- and 2-copy mice were suppressed in response to the high-salt diet compared with levels in mice fed the normal-salt diet. However, this diet did not suppress adrenal Aldo levels in 3- and 4-copy mice. The underlying mechanism may be related to the elevated sodium excretion and urine output that occurs with relatively lower blood pressure in 3- and 4-copy mice than in 2-copy mice; thus the inhibitory effect of the high-salt diet on adrenal Aldo levels was attenuated in 3- and 4-copy mice. Adrenal Aldo levels in 1- and 2-copy mice decreased to a similar level with 3- and 4-copy mice on a high-salt diet. Therefore, both the low-salt and high-salt data further confirmed that Npr1 gene copy number affects adrenal Aldo levels. Our findings further suggest that the adrenal gland is more sensitive to a low-salt diet than to a high-salt diet, which is consistent with the fact that the adrenal gland functions to retain salt to regulate blood volume homeostasis. Also, it is well known that a low-salt diet stimulates plasma ANG II and Aldo levels, whereas a high-salt diet inhibits them (18, 26).

We found that renal ANG II levels were decreased in 1-copy, 3-copy, and 4-copy mice compared with 2-copy mice. Interestingly, our previous studies have shown that renal ANG II levels increased in newborn pups of Npr1 gene-disrupted mice more than that of wild-type mice. However, renal ANG II levels decreased in both young (at 3 wk of age) as well as adult (at 16–26 wk of age) Npr1 gene-disrupted mice compared with wild-type mice (43). The mechanism may be due to the elevated blood pressure that decreases renal ANG II levels by inhibiting renin synthesis and release from the kidney juxtaglomerular cells as a negative feedback mechanism in Npr1 gene-disrupted mice (43). The significant changes in renal ANG II levels at different ages in Npr1 gene-disrupted mice indicated that renal ANG II levels may be regulated by ANP-NPRA signaling and also by additional factors, including salt diet, blood pressure, plasma and tissue RAS, and other hormonal milieu (2, 47, 49, 50). In 1-copy mice, adrenal ANG II and Aldo levels were higher than 2-copy mice. The mechanism might be related to increased adrenal renin levels in 1-copy mice compared with 2-copy mice (43). It may indicate that the adrenal RAS is mainly regulated by local tissue factors other than circulating factors. Variations in dietary sodium intake are closely associated with changes in renal renin and ANG II contents (28). In our study, the low-salt diet stimulated renal ANG II levels in 1-copy, 2-copy, 3-copy, and 4-copy mice compared with ANG II levels with the normal-salt diet. The high-salt diet suppressed renal ANG II levels in 1-copy and 2-copy mice, but not in 3-copy and 4-copy mice. These data also further confirmed that variations in dietary salt intake affect renal ANG II levels.

Interestingly, plasma ANG II levels decreased in 1-copy Npr1 mice compared with 2-copy mice, whereas plasma Aldo levels increased in 1-copy mice. The increased plasma Aldo levels may be due to elevation in the adrenal renin level in 1-copy mice compared with 2-copy mice, as reported previously (43). Elevated arterial blood pressure decreases the plasma ANG II level by inhibiting renin synthesis; the release of renin from the kidney juxtaglomerular cells is a negative feedback mechanism in 1-copy mice but not in 2-copy mice (43). On the other hand, decreased plasma ANG II levels in Npr1 gene-duplicated 3- and 4-copy mice compared with levels in 2-copy mice seem to be mainly a consequence of antagonism of ANP/NPRA signaling to the RAS. Plasma ANG II levels in different Npr1 gene copy mice indicated that the levels of ANG II in plasma might be regulated not only by NPRA signaling but also by factors such as arterial blood pressure and other hormonal factors. Elevations in renal ANG II levels cause reductions in renal function and sodium excretion that contribute to progressive hypertension and lead to renal and vascular injury (29). In the present study, the low-salt diet stimulated plasma ANG II and Aldo levels in 1-, 2-, 3-, and 4-copy Npr1 mice compared with levels found in the same mice given a normal-salt diet. However, the high-salt diet inhibited plasma ANG II and Aldo levels in 1- and 2-copy mice, but not in 3- and 4-copy mice. Increased sodium excretion and urine output with decreased arterial blood pressure in 3- and 4-copy mice might attenuate the inhibitory effect of the high-salt diet on plasma ANG II and Aldo levels (44). Our findings suggest that NPRA signaling has a protective effect on blood volume homeostasis and blood pressure regulation in Npr1 gene-duplicated mice compared with Npr1 gene-disrupted mice.

We further delineated the effect of Npr1 gene copy number on blood pressure in a gene-dose dependent manner with the three salt diets. Intriguingly, the high-salt diet caused an increase in blood pressure in 1- and 2-copy mice, but did not exert a significant effect on blood pressure in 3- and 4- copy mice. Previously, it has been reported that blood pressure was decreased in 4-copy mice on a high-salt diet compared with blood pressure in 4-copy mice fed a low-salt diet (31). Earlier studies have also suggested that the Npr1 gene, like the gene coding for ANP, may directly affect the sensitivity of blood pressure to salt (15, 25). Our present results with Npr1 gene-targeted mice provide evidence that NPRA signaling has a protective effect on blood pressure regulation in response to high salt. However, in another Npr1 gene knockout mouse model, neither a low- nor high-salt diet affected systemic blood pressure, although mice lacking the Npr1 gene had elevated systolic blood pressure and the authors concluded that hypertension is salt resistant in that model (20). Both of these mouse models focus on the Npr1 gene, but the detailed gene-targeting method is different (20, 30). In the model we used, the gene-targeting method involves replacing exon 1 and intron 1 of the Npr1 gene with a DNA fragment containing a neomycin-selectable marker (30). The other model uses simple insertion of a neomycin-resistance gene into exon 4 of the Npr1 gene (20). Although a similar degree of hypertension has been confirmed in that model, the heart, kidneys, and vasculature of the mutant mice were normal after histological examination at <5 mo of age (20, 30). The contradictory results in these models regarding the sensitivity of blood pressure to salt is likely to be related to the greater difficulty of observing small genetic effects in F2 vs. F1 mice (32). However, we cannot exclude the possibility that the contradictory results also involve the use of different gene-targeting methods. Our findings suggest that NPRA signaling has a protective effect against high-salt in Npr1 gene-duplicated mice compared with Npr1 gene-disrupted mice.

The results obtained in the present study provide evidence that adrenal ANG II and Aldo levels are increased in Npr1 gene-disrupted mice and, moreover, this increase appears to participate in elevating systemic blood pressure in mice with decreasing Npr1 gene-copy numbers. On the other hand, adrenal ANG II and Aldo levels are decreased in Npr1 gene-duplicated mice. A low-salt diet stimulated adrenal ANG II and Aldo levels in all Npr1 gene-targeted mice, whereas a high-salt diet suppressed adrenal ANG II and Aldo levels in Npr1 gene-disrupted mice and wild-type mice, but not in Npr1 gene-duplicated mice. Together, the present results clearly demonstrate the importance of ANP-NPRA signaling on the regulation of adrenal ANG II and Aldo levels. The interaction between ANP-NPRA signaling and local independent tissue ANG II and Aldo levels seems to be critical in the regulation of blood pressure and cardiovascular disease states.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the National Heart, Lung, and Blood Institute Grant HL-62147 and the Louisiana Board of Regents Health Excellence Fund.


   ACKNOWLEDGMENTS
 
We thank L. Charlene Binford and Gevoni Bolden for technical assistance and Kamala Pandey for assistance in preparing this manuscript. We are indebted to Dr. Oliver Smithies for providing initial breeding pairs of Npr1 gene-targeted mouse colonies. We also thank Dr. L. Gabriel Navar for stimulating discussion and Dr. Sudesh K. Srivastav for assistance in statistical analyses of the data. Our special thanks go to Dr. Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine, and Dr. Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory at the MD Anderson Cancer Center, for providing facilities during our displacement due to Hurricane Katrina.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Pandey, Dept. of Physiology, SL-39, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112 (e-mail: kpandey{at}tulane.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 45: 455–497, 1993.[Web of Science][Medline]
  2. Bassett M, White P, Rainey W. The regulation of aldosterone synthase expression. Mol Cell Endocrinol 217: 67–74, 2004.[CrossRef][Web of Science][Medline]
  3. Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev 70: 665–699, 1990.[Free Full Text]
  4. Chai S, Sexton P, Allen A, Figdor R, Mendelsohn F. In vitro autoradiographic localization of ANP receptors in rat kidney and adrenal gland. Am J Physiol Renal Fluid Electrolyte Physiol 250: F753–F757, 1986.[Abstract/Free Full Text]
  5. Chiou CY, Williams GH, Kifor I. Study of the rat adrenal renin-angiotensin system at a cellular level. J Clin Invest 96: 1375–1381, 1995.[Web of Science][Medline]
  6. de Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 230: 767–770, 1985.[Abstract/Free Full Text]
  7. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 28: 89–94, 1981.[CrossRef][Web of Science][Medline]
  8. Doi Y, Atarashi K, Franco-Saenz R, Mulrow PJ. Effects of changes in sodium or potassium balance, and nephrectomy, on adrenal renin and aldosterone concentrations. Hypertension 6: I124–I129, 1984.[Web of Science][Medline]
  9. Doi Y, Baba K, Doi Y, Franco-Saenz R, Mulrow PJ. Mechanisms by which nephrectomy stimulates adrenal renin. Hypertension 8: 997–1002, 1986.[Abstract/Free Full Text]
  10. Drewett JG, Garbers DL. The family of guanylyl cyclase receptors and their ligands. Endocr Rev 15: 135–162, 1994.[Abstract/Free Full Text]
  11. Ganguly A. Atrial natriuretic peptide-induced inhibition of aldosterone secretion: a quest for mediator(s). Am J Physiol Endocrinol Metab 263: E181–E194, 1992.[Abstract/Free Full Text]
  12. Garbers DL. Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands. Cell 71: 1–4, 1992.[CrossRef][Web of Science][Medline]
  13. Garg R, Oliver PM, Maeda N, Pandey KN. Genomic structure, organization, and promoter region analysis of murine guanylyl cyclase/atrial natriuretic peptide receptor-A gene. Gene 291: 123–133, 2002.[CrossRef][Web of Science][Medline]
  14. Garg R, Pandey KN. Regulation of guanylyl cyclase/natriuretic peptide receptor-A gene expression. Peptides 26: 1009–1023, 2005.[CrossRef][Web of Science][Medline]
  15. John SW, Krege JH, Oliver PM, Hagaman JR, Hodgin JB, Pang SC, Flynn TG, Smithies O. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 267: 679–681, 1995.[Abstract/Free Full Text]
  16. Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, Goeddel DV. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 252: 120–123, 1991.[Abstract/Free Full Text]
  17. Krege JH, Hodgin JB, Hagaman JR, Smithies O. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25: 1111–1115, 1995.[Abstract/Free Full Text]
  18. Laragh J, Sealey J. The renin-angiotensin-aldosterone system in hypertensive disorders: a key to two forms of arteriolar constriction and a possible clue to risk of vascular injury (heart attack and stroke and prognosis). In: Hypertension: Mechanisms, Diagnosis and Management. New York: Raven, 1990, p. 1329–1348.
  19. Levin ER, Gardner DG, Samson WK. Natriuretic peptides. N Engl J Med 339: 321–328, 1998.[Free Full Text]
  20. Lopez MJ, Wong SK, Kishimoto I, Dubois S, Mach V, Friesen J, Garbers DL, Beuve A. Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide. Nature 378: 65–68, 1995.[CrossRef][Medline]
  21. Lucas KA, Pitari GM, Dazerounian S, Ruiz-Stewart I, Park J, Schulz S, Chepenic KP, Waldman SA. Guanylyl cyclase and signaling by cyclic-GMP. Pharmacol Rev 52: 375–413, 2000.[Abstract/Free Full Text]
  22. Maack T. Role of atrial natriuretic factor in volume control. Kidney Int 49: 1732–1737, 1996.[Web of Science][Medline]
  23. Matsumoto M, Gotoh T, Suzuki-Kusaba M, Yoshida M, Hisa H, Satoh S. Effect of angiotensin II on aldosterone secretion in canine adrenal gland in situ. J Cardiovasc Pharmacol 36: 71–76, 2000.[CrossRef][Web of Science][Medline]
  24. Mazzocchi G, Malendowicz LK, Markowska A, Albertin G, Nussdorfer GG. Role of adrenal renin-angiotensin system in the control of aldosterone secretion in sodium-restricted rats. Am J Physiol Endocrinol Metab 278: E1027–E1030, 2000.[Abstract/Free Full Text]
  25. Melo LG, Veress AT, Chong CK, Pang SC, Flynn TG, Sonnenberg H. Salt-sensitive hypertension in ANP knockout mice: potential role of abnormal plasma renin activity. Am J Physiol Regul Integr Comp Physiol 274: R255–R261, 1998.[Abstract/Free Full Text]
  26. Morgan T. Interactions between sodium and angiotensin. Clin Exp Pharmacol Physiol 28: 1070–1073, 2001.[CrossRef][Web of Science][Medline]
  27. Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H. Brain natriuretic peptide as a novel adrenal hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest 87: 1402–1412, 1991.[Web of Science][Medline]
  28. Navar LG. The intrarenal renin-angiotensin system in hypertension. Kidney Int 65: 1522–1532, 2004.[CrossRef][Web of Science][Medline]
  29. Navar LG, Kobori H, Prieto-Carrasquero M. Intrarenal angiotensin II and hypertension. Curr Hypertens Rep 5: 135–143, 2003.[Web of Science][Medline]
  30. Oliver PM, Fox JE, Kim R, Rockman HA, Kim HS, Reddick RL, Pandey KN, Milgram SL, Smithies O, Maeda N. Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Pro Natl Acad Sci USA 94: 14730–14735, 1997.[Abstract/Free Full Text]
  31. Oliver PM, John SW, Purdy KE, Kim R, Maeda N, Goy MF, Smithies O. Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner. Proc Natl Acad Sci USA 95: 2547–2551, 1998.[Abstract/Free Full Text]
  32. Olson LJ, Ho BY, Cashdollar LW, Drewett JG. Functionally active catalytic domain is essential of guanylyl cyclase-linked receptor mediated inhibition of human aldosterone synthesis. Mol Pharmacol 54: 761–769, 1998.[Abstract/Free Full Text]
  33. Pandey KN. Biology of natriuretic peptides and their receptors. Peptides 26: 901–932, 2005.[CrossRef][Web of Science][Medline]
  34. Pandey KN. Physiology of the natriuretic peptides:gonadal function. In: Natriuretic Peptides in Health and Disease, edited by Samson WK and Levin E. Totowa, NJ: Humana, p. 171–191, 1997.
  35. Pandey KN. Vascular action natriuretic peptide receptor. In: Contemporary Endocrinology of the Vasculature, edited by Sowers JR. Totowa, NJ: Humana, p. 255–267, 1996.
  36. Pandey KN, Inagami T. Regulation of renin angiotensins by gonadotropic hormones in cultured murine Leydig tumor cells. Release of angiotensin but not renin. J Biol Chem 261: 3934–3938, 1986.[Abstract/Free Full Text]
  37. Pandey KN, Oliver PM, Maeda N, Smithies O. Hypertension associated with decreased testosterone levels in natriuretic peptide receptor-A gene-knockout and gene-duplicated mutant mouse models. Endocrinology 140: 5112–5119, 1999.[Abstract/Free Full Text]
  38. Pandey KN, Singh S. Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA. J Biol Chem 265: 12342–12348, 1990.[Abstract/Free Full Text]
  39. Robert MC, Helmy MS. Newly recognized components of the renin-angiotensin system: Potential roles in cardiovascular and renal regulation. Endocr Rev 24: 261–271, 2003.[Abstract/Free Full Text]
  40. Rosenzweig A, Seidman CE. Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 60: 229–255, 1991.[CrossRef][Web of Science][Medline]
  41. Schulz S, Singh S, Bekket RA, Singh G, Tubb DJ, Chin H, Garbers DL. The primary structure of a plasma membrane guanylyl cyclase demonstrates diversity within this new receptor family. Cell 58: 1155–1162, 1989.[CrossRef][Web of Science][Medline]
  42. Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 230: 3–30, 2002.[CrossRef][Web of Science][Medline]
  43. Shi SJ, Nguyen HT, Sharma GD, Navar LG, Pandey KN. Genetic disruption of atrial natriuretic peptide receptor-A alters renin and angiotensin II levels. Am J Physiol Renal Physiol 281: F665–F673, 2001.[Abstract/Free Full Text]
  44. Shi SJ, Vellaichamy E, Chin SY, Smithies O, Navar LG, Pandey KN. Natriuretic peptide receptor A mediates renal sodium excretory responses to blood volume expansion. Am J Physiol Renal Physiol 285: F694–F702, 2003.[Abstract/Free Full Text]
  45. Suga S, Nakao K, Hosoda K, Mukoyama M, Ogawa Y, Shirakami G, Arai H, Saito Y, Kambayashi Y, Inouye K., Imura H. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 130: 229–239, 1992.[Abstract/Free Full Text]
  46. Sugawara A, Nakao K, Morii N, Yamada T, Itoh H, Shiono S, Saito Y, Mukoyama M, Arai H, Nishimura K. Synthesis of atrial natriuretic polypeptide in human failing hearts. Evidence for altered processing of atrial natriuretic polypeptide precursor and augmented synthesis of beta-human ANP. J Clin Invest 81: 1962–1970, 1988.[Web of Science][Medline]
  47. Tremblay A, Parker KL, Lehoux JG. Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production. Endocrinology 130: 3152–3158, 1992.[Abstract/Free Full Text]
  48. Vellaichamy E, Khurana ML, Fink J, Pandey KN. Involvement of the NF-kappa B/matrix metalloproteinase pathway in cardiac fibrosis of mice lacking guanylyl cyclase/natriuretic peptide receptor A. J Biol Chem 280: 19230–19242, 2005.[Abstract/Free Full Text]
  49. Wu Z, Zheng W, Sandberg K. Estrogen regulates adrenal angiotensin type 1 receptors by modulating adrenal angiotensin levels. Endocrinology 144: 1350–1356, 2003.[Abstract/Free Full Text]
  50. Ye P, Kenyon C, MacKenzie S, Seckl J, Fraser R, Connell J, Davies E. Regulation of aldosterone synthase gene expression in the rat adrenal gland and central nervous system by sodium and angiotensin II. Endocrinology 144: 3321–3328, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F121    most recent
00478.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, D.
Right arrow Articles by Pandey, K. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, D.
Right arrow Articles by Pandey, K. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.