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Endocrinology Vol. 141, No. 1 325-332
Copyright © 2000 by The Endocrine Society

ARTICLES

Up-Regulation of Adrenal Cortical and Medullary Atrial Natriuretic Peptide and Gene Expression in Rats with Deoxycorticosterone Acetate-Salt Treatment1

Feng-Jie Lai, Shyi-Jang Shin, Yau-Jiunn Lee, Shiu-Ru Lin, Wen-Yi Jou and Juei-Hsiung Tsai

Graduate Institute of Medicine, Division of Endocrinology and Metabolism, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

Address all correspondence and requests for reprints to: Juei-Hsiung Tsai, M.D., Ph.D., Division of Endocrinology and Metabolism, Kaohsiung Medical University, 100 Shih-Chuan First Road, Kaohsiung 80708, Taiwan. E-mail: jhsiung{at}cc.kmc.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous study demonstrated that human adrenal medulla is a site of atrial natriuretic peptide (ANP) synthesis. To further evaluate the role of adrenal ANP in body fluid homeostasis, we investigated the changes in adrenal ANP in rats receiving deoxycorticosterone acetate (DOCA)-salt treatment. In situ hybridization and immunohistochemical study showed that adrenal ANP messenger RNA (mRNA) and ANP-like immunoreactivities (ANP-LI) were mainly localized in the zona glomerulosa and medulla of vehicle-treated rats. DOCA-salt treatment activated ANP mRNA and peptide expression in all adrenal zones, especially in the zona fasciculata/reticularis from 12 h to the entire 8-day study period. Using a semiquantitative RT-PCR technique, the relative quantities of ANP mRNA in the adrenals of the DOCA-salt-treated group were significantly increased from 1 to 8 days, whereas the adrenal weights of DOCA-salt-treated rats were significantly decreased from day 2 to day 8. Our results are the first to indicate that ANP is synthesized not only in the adrenal medulla but also in the adrenal cortex and their syntheses are markedly increased in DOCA-salt-treated rats. These results imply that adrenal ANP may participate in the intraadrenal regulation of adrenal function on water-electrolyte homeostasis in an autocrine or paracrine manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATRIAL NATRIURETIC peptide (ANP), a hormone originally identified in atrial cardiomyocytes, affects body fluid, salt excretion, and blood pressure through its concerted actions on various target organs, including vascular smooth muscle, kidney, and adrenal cortex (1, 2). Studies using immunohistochemical techniques to determine ANP and Northern blot analysis or RT-PCR to determine messenger RNA (mRNA) expression have demonstrated the ubiquitous distribution of ANP in various extraatrial tissues (3, 4, 5, 6, 7, 8), including the adrenal medulla (4, 5, 6, 7, 8). In our previous studies we found that the human adrenal medulla was a site of ANP synthesis, and adrenomedullary ANP synthesis was increased in patients with primary aldosteronism (7, 8). We also demonstrated that renal ANP synthesis was markedly enhanced in DOCA-salt-treated (9) and diabetic (10) rats. These findings indicate that the increase in adrenal and renal ANP synthesis may be responsive to volume expansion, just as atrial ANP synthesis and plasma ANP concentrations have been shown to be enhanced with increasing intravascular blood volume (11, 12, 13).

In adrenal cortical cells, exogenous ANP is a potent inhibitor of aldosterone synthesis and other steroidogenesis (14). In addition to the regulation of the adrenal function through the circulation, there is increasing evidence indicating that several intraadrenal mechanisms are involved in the regulation of adrenal function during physiological or pathophysiological processes of the adrenal gland (14). Therefore, it has been hypothesized that ANP is produced in the adrenal medulla and may be transported through the gland to act on the zona glomerulosa (7, 8). Although it is clear that certain neuropeptides may be transported in nerve fibers from the adrenal medulla to the cortex (15), there is no evidence to date that ANP is found in the nerves supplying the adrenal gland. Several investigations have demonstrated that adrenal cortical cells can produce a number of regulatory peptides as well as their receptors with similar distribution (16, 17). The three subtypes of ANP receptors have been found in both adrenal cortical and medullary cells (18). Therefore, we hypothesize that ANP can be synthesized in the adrenal cortex in addition to the adrenal medulla, and adrenal cortical ANP expression can be regulated in response to volume overloading. We designed the present study to observe the distribution of and changes in adrenal ANP mRNA and ANP in rats with or without deoxycorticosterone acetate plus 1% NaCl in drinking water (DOCA-salt) by in situ hybridization, RT-PCR, and immunohistochemical methods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal experiments
Male Wistar rats, 12–14 weeks of age and weighing 330–400 g, were housed individually in metabolic cages for 3 days before the start of the experiments and were divided into DOCA-salt-treated and vehicle-treated rats. DOCA-salt-treated rats were injected daily with a suspension of deoxycorticosterone acetate (25 mg/kg BW; Sigma, St. Louis, MO) dissolved in sesame oil for 0.5, 1, 2, 4, or 8 days (n = 3, 5, 5, 5, and 16, respectively) and were given free access to a 1% NaCl drinking solution. Body weight- and age-matched rats were similarly injected daily with vehicle (the same dose of sesame oil) for 0.5, 1, 2, 4, or 8 days (n = 3, 4, 4, 4, and 15, respectively) and were given tap water to drink. All rats were fed normal rat laboratory chow. Body weight was recorded at the beginning and end of the experiment. Systolic blood pressure in the 8-day treated group was measured at least 12 h before decapitation in the conscious restrained rats by tail cuff sphygmomanometry (KN-210, Natsume, Tokyo, Japan). The blood pressure value for each rat was calculated as the average of 10 separated measurements. Rats were killed at the end of the experiment by decapitation to collect blood and tissues. Truncal blood was collected in prechilled tubes containing EDTA (1 mg/ml) and aprotinin (500 kIU/ml), and plasma was stored at -20 C until assay. An abdominal incision was made for removal of the adrenal glands. The periadrenal fat was carefully dissected, and the glands were weighed in an analytical scale (Sartorius, Brinkmann Instruments, Inc., Westbury, NY; precision, 0.1 mg). The right adrenal of each animal was frozen and stored at -70 C for RNA extraction; the left adrenal was bisected and fixed in 4% paraformaldehyde for over 10 h, then paraffin embedded, following standard procedures. The adrenals of the vehicle- and DOCA-salt-treated rats were embedded within the same paraffin block and cut into 4-µm sections. We compared the relative amounts of adrenal ANP mRNA or ANP-LI of vehicle- and DOCA-salt-treated rats on the same slide by in situ hybridization or immunohistochemical study to avoid discrepancies due to varying thickness and different processing times. The animal studies were approval by the animal care and treatment committee of our institution.

Plasma samples
ANP in plasma samples was determined using a specific RIA kit (Peninsula Laboratories, Inc., Belmont, CA) after extraction as in our previous report (7, 9). Briefly, 3 ml plasma were passed through Sep-Pak C18 cartridges (Waters Corp., Milford, MA) and eluted with 5 ml 60% acetonitrile in 0.1% trifluoroacetic acid. The eluate was lyophilized and reconstituted for RIA. PRA was measured by enzymatic incubation of plasma at 37 C and pH 6.4. The angiotensin I generated during the incubation step was quantified using a RIA kit (DuPont-NEN, Boston, MA). The concentrations of plasma sodium were determined in an automatic analyzer (Nova Biochemical, Newton, MA).

Isolation of total RNA
Total RNA was isolated from the right adrenal gland by a modified guanidium isothiocyanate method (19). To remove contaminating genomic DNA, RNA samples were incubated with 1 U ribonuclease-free deoxyribonuclease (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min at 37 C in 50 µl of a buffer containing 40 mmol/liter Tris-HCl (pH 7.9), 10 mmol/liter NaCl, 6 mmol/liter MgCl2, and 10 mmol/liter CaCl2. After deoxyribonuclease treatment, the samples were reextracted by the modified guanidium isothiocyanate method. The integrity of the RNA was assessed by formaldehyde-agarose gel electrophoresis followed by ethidium bromide staining, and the quantity was determined by absorbance at 260 nm.

RT
Two micrograms of total RNA from right adrenals were reverse transcribed by incubation with a 20-µl RT mixture containing 20 pmol oligo(deoxythymidine)18 primer, 50 mM Tris-HCl (pH 8.3), 75 mM deoxy-NTPs, and 50 U Moloney murine leukemia virus reverse transcriptase (Stratagene, Palo Alto, CA) at 37 C for 2 h. The reverse transcriptase was inactivated by heating for 5 min at 94 C.

PCR amplification
Sequences for rat ANP and ß-actin were obtained from GenBank and used to design the primer pairs (Table 1Go). Primers were chosen to contain a GC content of 40–60% and to have a genomic structure separated by an intron. Each 50 µl PCR contained 5 µl reverse transcriptase reaction mixture, 1 x PCR buffer [50 mM Tris-HCl (pH 9.1), 14 mM (NH4)2SO4, and 1.75 mM MgCl2], 0.2 mM of each deoxy-NTP, 2 U Taq polymerase (Quality Systems, Inc., Taipei, Republic of China), and 0.1 µmol/liter of each specific primer pair. After an initial heating step at 92 C for 1 min, the amplification cycles were 30 sec at 94 C, 30 sec at 60 C, and 60 sec at 72 C, ending with 72 C for 7 min in a thermocycler (model 9600, Perkin-Elmer Corp., Norwalk, CT). Preliminary experiments were conducted to determine the optimal number of PCR cycles for ANP and ß-actin. The cycle number for the amplification of ANP and ß-actin complementary DNA (cDNA) was repeated from 32–42 times and from 12–22 times, respectively. The cycle selected for PCR amplification was 34 times for ANP and 14 times for ß-actin.


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  		Table 1. PCR primer name, sequence, and product size

 
Southern blot analysis
The ANP and ß-actin PCR products were then electrophoresed on 1.5% agarose gels and transferred to nylon membranes (Schleicher & Schuell, Inc., Dassel, Germany). The blots were hybridized with a digoxigenin-labeled, random primed, 436-bp rat ANP cDNA prepared from PCR cloning of the rat atria extracts for 16 h at 42 C according to the standard technique (Roche Molecular Biochemicals). After each hybridization, the blot was washed twice in a solution containing 0.1% SDS and 2 x SSC (standard saline citrate) for 15 min at room temperature, then twice in 0.1% SDS and 0.1 x SSC at 68 C. Immunodetection steps and chemiluminescent reaction with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) were carried out according to the manufacturer’s instructions provided with the DIG Luminescent Detection Kit (Roche Molecular Biochemicals). Blots were exposed to Kodak BioMax MR-1 film (Eastman Kodak Co., Rochester, NY) at room temperature. A digoxigenin-labeled probe of ß-actin, used as an internal control, was also made using the primer extension method. After luminescent exposure, the x-ray film was scanned by a laser densitometer (Molecular Dynamics, Inc., Sunnyvale, CA), and the data were analyzed by M.D. ImageQuant software (version 3.22, Molecular Dynamics, Inc.). The value obtained for each ANP PCR product was normalized by the ß-actin cDNA PCR product.

Complementary RNA (cRNA) probes
To generate T7 promoter-tailed ANP DNA templates for in vitro transcription for ANP cRNA probes, two successive PCR reactions were performed as described by Cone et al. (20). In the first PCR reaction, 2 separate amplifications for 30 cycles under standard conditions were performed using the ANP (sense)/T7+ANP (antisense) or T7+ANP (sense)/ANP (antisense) primer pairs; these sequences are listed in Table 1Go. For each primer pair, 1 primer had no additional nucleotides (ANP sequence only), and the other primer was tailed with the 3'-end of the T7 promoter. The 2 reaction products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. The first PCR products were purified with a purification kit (Roche Molecular Biochemicals). Five microliters of a 1:100 dilution of the first PCR products were reamplified using 1 ANP primer as used in the first round of PCR and another composite primer (universal T7; Table 1Go) to extend and complete the full-length 23-bp T7 promoter. The second PCR products were electrophoresed on a 1.5% agarose gel, and the resulting bands were excised, purified using the Qiaex II Gel extraction kit (QIAGEN AG, Berne, Switzerland), and quantified by spectrophotometry.

Digoxigenin-labeled antisense and sense ANP cRNA probes were separately transcribed and labeled in a reaction containing the respective T7 promoter-tailed ANP PCR product (1 µg), T7 polymerase, and digoxigenin-11-UTP using the Dig RNA Labeling Kit (Roche Molecular Biochemicals) according to the manufacturer’s instructions.

In situ hybridization
Sections were pretreated with microwave heating as described by Lan et al. (21). Briefly, after deparaffinization and rehydration, sections were placed in a universal slide kit filled with 0.01 M sodium citrate buffer (300 ml; pH 6.0). Slides were heated twice for periods of 5 min at the maximal power setting (720 watts) of the microwave oven with 140–145 sec of boiling time. After microwave pretreatment, sections were washed in 2 x SSC twice for 5 min each time and then prehybridized with 150 µl hybridization buffer containing 50% deionized formamide, 4 x SSC, 2 x Denhardt’s solution, 1 mg/ml salmon sperm DNA (Roche Molecular Biochemicals), and 1 mg/ml yeast transfer RNA for 1 h at 42 C. After washing in 2 x SSC, sections were incubated with 100 µl of the hybridization buffer containing 300–800 ng/ml denatured digoxigenin-labeled probe for 3 h at 42 C. After hybridization the sections were washed twice with 2 x SSC for 5 min at room temperature, followed by 0.1 x SSC for 30 min at 42 C. Digoxigenin-labeled RNA hybrids were detected using an enzyme-linked immunoassay kit (Roche Molecular Biochemicals). After immersion in 1.5% blocking solution, the slides were exposed to antidigoxigenin alkaline phosphatase conjugate diluted 1:1000 for 30 min. The hybrids were visualized as purple/black precipitates by subsequent alkaline phosphatase-chloro-3-indolyl phosphate and nitro blue tetrazolium.

Immunohistochemistry
Sections were pretreated with microwave heating as described above. After microwave treatment, sections were washed in PBS and incubated with 1% BSA for 30 min to block nonspecific staining. Sections were drained and incubated for 3 h at room temperature in a humidity chamber with the respective rabbit antirat primary antibody for {alpha}ANP (1:1000 dilution) diluted with antibody diluent (DAKO Corp., Glostrup, Denmark). The primary antibody was purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). After washing in PBS, endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 20 min, followed by sequential 10-min incubations with biotinylated link antibody and peroxidase-labeled streptavidin (DAKO Corp., LSAB 2 kit). Staining was completed after incubation with Vector VIP substrate-chromogen solution, then counterstained with methyl green (Vector Laboratories, Inc., Burlingame, CA), and mounted in aqueous mounting medium.

Statistical analysis
The data are expressed as the mean ± SEM. To test the difference between vehicle-treated and DOCA-salt-treated rats, unpaired Student’s t test was performed. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The characteristics of vehicle-treated and DOCA-salt-treated groups on the eighth study day are shown in Table 2Go. There was no significant difference in initial body weight between the 8-day vehicle-treated and 8-day DOCA-salt-treated rats. All animals gained weight over the 8-day course of the experiment. However, the weight gain of the DOCA-salt-treated rats was significantly less than that of the vehicle-treated rats. Tail cuff systolic blood pressures were not different between the two groups. Plasma ANP level was significantly elevated in DOCA-salt-treated rats compared with that in vehicle-treated rats. In contrast, PRA fell to almost undetectable levels in DOCA-salt-treated rats. Plasma sodium levels rose significantly in DOCA-salt-treated rats compared with those in vehicle-treated rats. Table 3Go shows that the adrenal weights of DOCA-salt-treated rats were significantly lighter than those of vehicle-treated animals from 2 days for the entire 8 days of the experiment.


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  		Table 2. Data for vehicle-treated and DOCA-salt-treated rats on the eighth study day

 

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  		Table 3. Comparison of adrenal weight/body weight (micrograms per g) in vehicle-treated and DOCA-salt-treated rats on days 1, 2, 4, and 8

 
In situ hybridization
We determined directly how ANP mRNA was distributed by in situ hybridization of adrenal sections from vehicle-treated and DOCA-salt-treated rats using a specific cRNA probe for ANP (Fig. 1Go, A–C; Fig. 2Go, A–D; and Table 4Go). In vehicle-treated rats, the signal of adrenal ANP mRNA was detected mainly in the zona glomerulosa and medulla (Fig. 1AGo), although some expression was also detected in the outer region of zona fasciculata (Fig. 2AGo). In DOCA-salt-treated rats, the density of ANP mRNA hybridization increased slightly in the outer region of the zona fasciculata at 12 h (Fig. 2BGo) and remained elevated for 8 days in all adrenal zones (Fig. 2Go, C and D; Fig. 1BGo; and Table 4Go) relative to that in vehicle-treated controls. Positive staining was restricted to the cytoplasm of parenchymal cells (Fig. 1CGo). We detected no signal using a sense cRNA probe (Fig. 1DGo).



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  		Figure 1. Representative photomicrographs show in situ hybridization of ANP mRNA (upper panel) and immunostaining of ANP (low panel) in the adrenal gland of 8-day vehicle-treated (A and E) and 8-day DOCA-salt-treated (B and F) rats. D shows the method control, in which the ANP antisense is replaced by a sense probe. H also shows the method control, in which the ANP antibody is replaced by normal rabbit serum. zg, zona glomerulosa; zf, zona fasciculata; zr, zona reticularis; m, medulla. Positive staining of ANP mRNA and ANP-LI was observed mainly in the zona glomerulosa and medulla in vehicle-treated rats (A and E). After 8-day DOCA-salt treatment, both adrenal ANP mRNA and ANP-LI staining were increased in the zona glomerulosa, zona fasciculata, and medulla, and there was faint reactivity in the zona reticularis (B and F). C and G show high power magnification of the outer cortical regions in B and F, respectively; positive staining is restricted to the cytoplasm of parenchymal cells. Method controls (D and H) are negative. Bar, 100 µm.

 


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  		Figure 2. Representative photomicrographs show in situ hybridization of ANP mRNA (upper panel) and immunostaining of ANP (low panel) in the adrenal glands at 0 h, 12 h, 1 day, and 4 days after DOCA-salt treatment. zg, Zona glomerulosa; zf, zona fasciculata; zr, zona reticularis; m, medulla. In control rats, positive staining of ANP mRNA and ANP-LI was observed mainly in the medulla and zona glomerulosa, although some expression was also detected in the zona fasciculata (A and E). After 12-h DOCA-salt treatment, slightly increased expression of ANP mRNA and ANP-LI was observed in the outer region of the zona fasciculata (B and F). The staining area and intensity of ANP mRNA and ANP-LI were progressively enhanced in the adrenal cortex at 1 day (C and G) and 4 days (D and H) after DOCA-salt treatment. Bar, 50 µm.

 

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  		Table 4. The zonal distribution of adrenal ANP mRNA and ANP-LI by in situ hybridization and immunohistochemical study from 8-day vehicle-treated and 8-day DOCA-salt-treated rats

 
Immunohistochemistry
Immunohistochemical localization of the ANP-LI in the adrenal gland was determined in both groups (Fig. 1Go, E–G; Fig. 2Go, E–H; and Table 4Go). The zonal distribution of adrenal ANP-LI was well in accordance with that of adrenal ANP mRNA by in situ hybridization. In vehicle-treated rats, ANP-LI staining was strong in the adrenal medulla, but only faint and inhomogeneous in the zona glomerulosa, with a slight preference for the subcapsular cell layer (Figs. 1EGo and 2EGo). After DOCA-salt treatment, the adrenal ANP-LI was enhanced in the outer region of the zona fasciculata at 12 h (Fig. 2FGo), and the staining area and/or intensity was further increased in the zona glomerulosa, zona fasciculata/reticularis, and medulla for 8 days (Fig. 2Go, G and H; Fig. 1FGo; and Table 4Go). Positive staining was restricted to the cytoplasm of parenchymal cells as fine granules and was undetectable in such nonparenchymal cells as capsular fibroblasts, sinusoidal lining cells, and endothelial cells (Fig. 1GGo). Control studies with primary antibodies that had been preabsorbed with the appropriate purified ANP or replacement of primary antibodies by normal rabbit serum did not produce positive stains (Fig. 1HGo).

ANP mRNA in adrenal gland
RT-PCR coupled with Southern blot analysis revealed a 436-bp product from rat adrenals and heart tissue RNA extracts (Fig. 3Go). As the ANP primer sets used in the current study span a 104-bp intron, the 436-bp product amplified by these primers could not be of genomic origin (Fig. 3Go). To determine the relative changes in tissue ANP mRNA expression, the yield of ANP PCR products was normalized to the amount of ß-actin cDNA amplified from the tissue samples. This method has been used in our previous studies (9, 10, 22) and has been able to measure a small change in the relative amounts of a specific mRNA with reasonable accuracy. The amplification cycles of the PCR procedure were evaluated. Figure 4Go shows the results of different PCR cycles for adrenal ANP cDNA and ß-actin cDNA. The following PCR analysis was performed only during the exponential phase.



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  		Figure 3. A, Photograph of ethidium bromide-stained agarose gel shows ANP cDNA of right atrium (lane 1) and adrenal gland (lane 2), a negative control (lane 3), and rat ANP genomic DNA (lane 4) by PCR. As described in Materials and Methods, cDNA (436 bp) and genomic DNA (540 bp) were amplified. The expression of the adrenal ANP gene was too low to be detected in ethidium bromide-stained agarose gel. M, Mol wt markers (Marker VI, Roche Molecular Biochemicals). B, Southern blots of the same samples as those in A were hybridized with a digoxigenin-labeled rat ANP-cDNA probe. The adrenal ANP gene was detected by RT-PCR followed by Southern blot analysis.

 


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  		Figure 4. Determination of compatible PCR cycle numbers for adrenal ANP cDNA and ß-actin cDNA by Southern blot analysis. A, Adrenal ANP cDNA were amplified from 32 to 42 cycles. Densitometric analysis showed that the amplification was within the exponential phase when the PCR cycle used for amplification was under 38 for adrenal ANP cDNA. B, Adrenal ß-actin cDNA were amplified from 12–22 cycles; according to the densitometric analysis, the cycle number selected for PCR amplification for ß-actin cDNA was 14 and was in the exponential phase of PCR amplification.

 
Figure 5AGo shows the results of RT-PCR followed by Southern blot analysis for amplification of ANP and ß-actin mRNA in the adrenals from vehicle-treated and DOCA-salt-treated rats on day 8. The relative ratios of the densitometry measures of the PCR products for ANP and ß-actin are shown in Fig. 5BGo. Significantly increased ANP mRNA levels were observed in the adrenals of DOCA-salt-treated rats from 1–8 days.



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  		Figure 5. A, The amplification of ANP and ß-actin mRNA by RT-PCR followed by Southern blot analysis in the adrenal glands from 4 representative 8-day vehicle-treated (Control) and 5 representative 8-day DOCA-salt-treated (DOCA-salt) rats. Each lane represents an individual rat. B, Relative tissue concentration of ANP mRNA in adrenal glands at 1, 2, 4, and 8 days after vehicle or DOCA-salt treatment, as determined by RT-PCR followed by Southern blot. Relative ratios (mean ± SEM) of the densitometry readings of ANP and ß-actin mRNA in the adrenal glands from 4 vehicle-treated and 5 DOCA-salt-treated rats on days 1, 2, and 4, and from 15 vehicle-treated and 16 DOCA-salt-treated rats on day 8. **, P < 0.01; ***, P < 0.001 (compared with vehicle-treated group, by Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present set of experiments applied in situ hybridization and immunohistochemical methods to assess whether the rat adrenal cortex has the capacity to synthesize ANP and to increase the expression of ANP in response to DOCA-salt treatment. The results clearly demonstrate that rat adrenal cortical cells have the capacity to produce ANP. Cells expressing ANP were found predominantly in the zona glomerulosa and medulla in control rats and additionally in the zona fasciculata/reticularis after DOCA-salt treatment. These findings indicate that ANP expression in the adrenal cortex was dynamically regulated and modulation of ANP expression in the adrenal gland might also be a mechanism for regulation of steroidogenesis.

A previous in situ hybridization study with an oligonucleotide ANP probe reported the presence of ANP mRNA in only about 15% of rat adrenal medullary cells, with no hybridization in the cortex (23). In our study, not only were all adrenal medulla cells hybridized, but also zona glomerulosa cells of vehicle-treated adrenals were hybridized. There were methodological differences in our experiments. We used paraffin sections with microwave treatment instead of frozen sections without microwave treatment, and cRNA probes instead of oligonucleotide probes. The value of microwave treatment for improving the sensitivity and reliability of mRNA by in situ hybridization or antigen detection by immunohistochemical method has been described (21, 24). Furthermore, the use of cRNA probes has been shown to produce hybridization of higher specificity and sensitivity (25, 26). We speculate that the methodological improvements may account for the different results.

The increase in plasma ANP levels and the decrease in PRA in DOCA-salt-treated rats were consistent with our previous findings and were thought to be mainly due to the expansion of extracellular fluid (9, 27). The elevation of circulating ANP can reduce cardiac output and systemic arterial blood pressure (28). However, with a low level of ANP gene expression in the adrenal gland, it is still difficult to determine whether adrenal-synthesized ANP really contributes to the circulation pool and acts in an endocrine manner to regulate water-electrolyte homeostasis.

In contrast with the large number of hormones and factors that stimulate adrenal corticoid secretion, only a few regulatory factors have been shown to inhibit adrenal steroidogenesis. ANP, a peptide of cardiac origin with profound vasorelaxant, diuretic, and natriuretic effects, directly inhibits aldosterone, cortisol, and dehydroepiandrosterone in cultured human adrenal cells (29) by acting through a specific receptor (30). The existence of ANP in the adrenal cortex suggests that this peptide might constitute a local endogenous ligand of intraadrenal origin for these receptors. Although the levels of ANP mRNA in the adrenal are low relative to those in a normal rat atrium (31), local synthesis of this peptide may produce high concentrations in close proximity to receptors and may be a confounding factor affecting the correlation between changes in circulating ANP and the observed biological effects. Therefore, the possibility cannot be excluded that ANP may affect adrenal function in a paracrine and/or endocrine manner as well as in an autocrine fashion.

The results showed that treatment with DOCA-salt increase ANP mRNA and peptides within the initial first day, and these levels are continuously enhanced in all zones of the adrenal gland especially in the zona fasciculata/reticularis. The change in ANP gene expression resulted from increases in both staining area and staining intensity. This may be of pathophysiological importance, as stimulation of ANP production by sodium intake and extra-DOCA injection can reduce aldosterone secretion and increased sodium excretion, associated with the mineralocorticoid escape phenomenon (32, 33). The recruitment of ANP expression cells may represent a novel mechanism for modifying the adrenal function response to DOCA-salt treatment. However, what factors are involved in this up-regulation of expression of ANP gene in the adrenal gland of rats? It is well known that DOCA-salt alters plasma electrolyte concentrations and levels of neurohormonal factors (34). It is possible that DOCA-salt-induced changes in these parameters may have direct or indirect effects on adrenal ANP expression. These subjects are worthy of further investigation.

We have often observed a reduction in the size of the adrenal glands in DOCA-salt-treated rats. It is well known that ANP possesses growth inhibitory properties. In the kidney and blood vessel, several investigations have demonstrated that ANP can inhibit cell proliferation and hypertrophy (35). Similarly, ANP has been observed to inhibit growth in the adrenal glands, especially in the zona glomerulosa (36). In this study, a time-course comparison with DOCA-salt treatment provided the opportunity to define the possible relationships between adrenal ANP expression and the development of adrenal atrophy induced by mineralocorticoid and salt excess. Findings obtained during the time course of the experiment showed that significant adrenal atrophy occurred on the second day of DOCA-salt treatment, whereas significant adrenal ANP elevation occurred within the first day of treatment, suggesting that adrenal ANP expression may be a trigger for the adrenal atrophy in this model. However, adrenal weights are influenced by many factors, such as ACTH (37), angiotensin I (38), and angiotensin II (39). To prove that ANP has some effect on adrenal atrophy in the DOCA-salt model, further investigation is required, such as chronically administering HS-142–1, a specific antagonist of the guanylate cyclase-coupled natriuretic peptide receptors (40), to DOCA-salt rats to determine whether adrenal weights are influenced.

In summary, the present study shows that the adrenal cortex, in addition to the adrenal medulla, is a site of ANP synthesis. In a volume expansion state such as DOCA-salt treatment, adrenal cortical and medullary syntheses of ANP are markedly enhanced, which indicates that the intraadrenal ANP system may play a role in water-electrolyte homeostasis by regulating adrenal functions in an autocrine/paracrine manner. The present study has also suggested a possible relation between the adrenal atrophy and adrenal ANP expression of DOCA-salt-treated rats. The precise mechanisms of the regulation of ANP expression in the adrenal gland remain to be determined.


   Acknowledgments
 
We thank Dr. Chiang-Shin Liu, Dr. Kun-Bow Tsai, and Mr. Chu-Ho Huang for their excellent technical assistance.


   Footnotes
 
1 This work was supported by grants (NSC-87–2314-B037–020-M21) from National Science Council, Taiwan. Back

Received June 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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