Although originally isolated from the atria of the heart (1,2) atrial natriuretic factor (ANF) also is found in several tissues including aortic tissue. Given the biologic properties of ANF, it may be expected that in these locations, this hormone acts as a modulator of endothelial function and of vascular growth. The latter includes the consensus function of this hormone as modulator of the renin-angiotensin system (RAS) in its short-term vasoconstrictive action (3) as well as in its long-term effect promoting vascular smooth-muscle cell growth (4). Vascular smooth muscle is one of the major tissue targets of ANF, in which it induces vasorelaxation and inhibitory regulation of proliferation and hypertrophy of aortic vascular smooth-muscle cells (4,5). By immunocytochemistry, ANF has been localized in the adventitia of the aortic arch (6), whereas blot-hybridization analysis demonstrated very low concentrations of ANF messenger RNA (mRNA) in the aortic arch and abdominal aorta in rats (6). By radioimmunoassay, both the N-terminus and C-terminus of the ANF prohormone were reported in the rat aorta (7). These reports suggest that ANF is synthesized in the aorta, but the concentration of specific message is below that needed to reliably quantify ANF mRNA with previously available technology.
We previously found (8; T. Ogawa, unpublished observations), by using the experimental hypertensive models described here, that the regulation of left atrial ANF mRNA is related to volume load, that the regulation of ventricular ANF mRNA is related to pressure load, and that the regulation of renal ANF mRNA levels is related to the RAS, especially plasma angiotensin II (AII) levels and renal angiotensin-converting enzyme (ACE) mRNA levels. These studies also demonstrated that although increased ANF gene expression is a marker of hypertrophy, upregulation of ANF gene expression is not necessarily associated with hypertrophy. Therefore, in defining the molecular events associated with cell hypertrophy, it is useful to compare more than one molecular marker of hypertrophy. For this reason in these studies, we determined α1 and α2 Na+K+ adenosine triphosphatase (ATPase) mRNA levels as a molecular marker of pressure overload in aorta. Na+K+-ATPase is a heterodimer enzyme consisting of an α subunit possessing catalytic activity and a β subunit of unknown function. The α subunit contains three isoforms (α1, α2, and α3; 9). It has been proposed that reduced activity of the Na+K+-ATPase activity in vascular smooth muscle would result in a decreased sodium gradient and decreased calcium extrusion via the Na+-Ca2+ exchanger. The resulting increased cellular Ca2+ content would enhance the vascular smooth-muscle tone and increase peripheral vascular smooth-muscle resistance, leading to hypertension. Herrera et al. (10,11) showed the downregulation of α2 Na+K+-ATPase mRNA levels in the aorta of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and in AII-infused hypertensive rats.
In this study, we investigated the effect of hypertension on aortic ANF gene expression as reflected by measurement of steady-state ANF mRNA levels in aortic tissue by using a recently developed quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR; 12), which allows reliable measurement of low transcript concentration. As an independent measure of cell hypertrophy, we determined Na+K+-ATPase isoform switch and parameters that are known to be important in the regulation of ANF gene expression, including blood pressure, and of RAS components. Evidence was obtained showing that, in contrast to our previous findings in hypertensive heart and kidney (8; T. Ogawa, unpublished observations), the regulation of ANF mRNA levels in aortic tissue is largely independent of pressure load, volume load, and plasma or tissue RAS.
Male Sprague-Dawley rats, weighing 100-125 g, were separated into (a) control, (b) DOCA, (c) salt, and (d) DOCA-salt groups. Treatment of animals was conducted by following institutional guidelines. The DOCA and the DOCA-salt groups rats were injected subcutaneously with a suspension of DOCA (30 mg/kg; Sigma Chemical, St. Louis, MO, U.S.A.) dissolved in sesame oil once a week. The salt and the DOCA-salt groups rats were given free access to 1% NaCl drinking solution during the experiment. Five weeks later, blood pressure was measured by tail sphygmomanometry, and the rats were killed by decapitation. Aorta samples were obtained distal to the aortic arch down to the bifurcation of the femoral arteries. After weighing, the tissue samples were quickly wrapped in aluminum foil and snap frozen in liquid nitrogen.
Adult male Sprague-Dawley rats weighing 270-280 g were fasted for 12 h before surgery. After the anesthesia (induced by halothane/nitrous oxide/oxygen), the abdomen was opened by a cut parallel to the linea alba. The abdominal aorta above the kidney was exposed, and a cannula was placed longitudinal to the aorta, and the two tied together. The cannula was pulled out, leaving the aorta constricted to the outer diameter of the cannula. Tetracycline was applied to the exposed field, and the abdomen was closed by clipping. Sham-operated animals were subjected to the same procedure without aortic banding. The rats were separated into five groups as follows: (a) control, (b) sham operated, (c) aortic banded, (d) aortic banded with high-dose ramipril (1 mg/kg), and (e) aortic banded with low-dose ramipril (10 μg/kg). Two protocols were followed:
- Ramipril was administered by daily oral gavage for 6 weeks to rats immediately after the aortic-banding operation (prevention experiment).
- Ramipril was started 6 weeks after the aortic banding and was continued for 6 weeks (regression experiment).
Ramipril dosage was adjusted weekly according to body weight. At the end of the treatment period, the animals were instrumented for measurement of carotid blood pressure, as tail sphygmomanometry is not feasible because of the blood pressure decrease distal to the banding of the aorta. Both methods of pressure measurement give comparable results (13,14). Aorta samples were obtained distal of the aortic arch down to the site of the banding. After weighing, the tissue samples were quickly wrapped in aluminum foil and snap frozen in liquid nitrogen.
Total RNA extraction and Northern blot analysis
Total RNA extraction and Northern blot analysis were performed as previously described (8) with the following 32P-labeled probes: a 332-bp EcoRI/Pst I fragment of the rat α1 Na+K+-ATPase cDNA (9) a 381-bp EcoRI/Pst I fragment of the rat α2 Na+K+-ATPase cDNA (9), a 1.6-kb EcoRI/HindIII fragment of the rat angiotensinogen cDNA (15), and a 5-kb SalI/EcoRI fragment of mouse 28S rRNA cDNA. Autoradiographs were scanned by using an Ultrascan XL laser densitometer (LKB Produckter, Uppsala, Sweden) and LKB 2400 Gelscan XL software package. Values obtained for each mRNA were normalized to 28S rRNA, which served as internal control to correct for differences in the amount of RNA applied and transfer efficiency.
Quantitative competitive RT-PCR
Details of this technique, including the preparation of the competitor recombinant ANF RNA, have been previously described (12). For each sample, aliquots of total RNA (5 μg) were used to prepare a dilution series to which competitor RNA was added. After PCR, aliquots (5 μl) of the product were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. Photograph negatives of the gels were scanned by laser densitometry and Gelscan XL 2400 software package. The ratio of the density of the competitor RNA to the target RNA was plotted against the amount of the competitor RNA added to each reaction. The competitor RNA amount, where this ratio is equal to 1, represents the amount of ANF mRNA present in the initial RNA sample.
All data were expressed as mean ± SEM, and a level of p < 0.05 was considered significant. Analysis of variance (ANOVA) was performed to determine statistical differences among multiple groups. When significance was obtained by ANOVA, Fisher's Least Squares Differences post hoc analysis was used to determine pairwise differences.
Systolic blood pressure, body weight, and aorta weight
In the DOCA-salt experiment, the systolic blood pressures of the rats treated with DOCA-salt were significantly higher than those of the control, DOCA-treated and salt-treated rats (Table 1). The systolic blood pressures of the DOCA-treated rats and salt-treated rats were not elevated compared with those of the control rats. The aortic-weight/body-weight ratios of the rats treated with DOCA alone, salt alone, or DOCA-salt were significantly higher than those of the control rats. In the aortic-banding experiments, the systolic blood pressures of the banded rats were significantly higher than those of the control and sham-operated rats. The systolic blood pressures of the banded rats treated with high-dose ramipril were normalized, whereas those of the banded rats treated with low-dose ramipril were similar to the levels of the banded rats. In the aortic-banding prevention experiment, the aortic-weight/body-weight ratio was similar among groups. In the aortic-banding regression experiment, the aortic-weight/body-weight ratio of the banded rats was significantly increased compared with that of the control rats. The ratios for the banded rats treated with either high-dose or low-dose ramipril were lower than those of the banded rats and similar to those of the control or sham-operated rats (Table 2).
Relative α1 and α2 Na+K+-ATPase mRNA levels
The α1 Na+K+-ATPase cDNA hybridized to a single 3.7-kb band, and α2 Na+K+-ATPase cDNA detected two 3.4- and 5.3-kb bands (Fig. 1). In the DOCA-salt experiment, relative α1 and α2 Na+K+-ATPase mRNA levels were similar among groups (Fig. 2). In the aortic-banding experiment, whereas the α1 mRNA levels were similar among groups, the α2 mRNA levels of the banded rats were significantly decreased compared with the control and sham-operated rats. Those parameters in the banded rats treated with high-dose ramipril returned to the control and sham-operated levels. The α2 Na+K+-ATPase mRNA levels in the banded rats treated with low-dose ramipril were similar to the control and sham-operated rats' levels (prevention experiment) or lower than those of the control and sham-operated rats' levels (regression experiment).
Angiotensinogen mRNA levels
In the DOCA-salt experiment, the angiotensinogen mRNA levels of the DOCA alone, salt alone, and DOCA-salt rats were significantly higher than those of the control rats (Fig. 3). In the aortic-banding prevention experiments, angiotensinogen mRNA levels of the banded rats were lower than those of the control or sham-operated rats. In the banded rats treated with high-dose ramipril, those parameters were higher than those of the control, sham-operated, and banded rats. In the banded rats treated with low-dose ramipril, those parameters were lower than those of the control, sham-operated, and banded rats treated with high-dose ramipril. In the regression experiment, the angiotensinogen mRNA levels showed levels similar to those of the prevention experiment, but the differences between the groups were less prominent than in the prevention experiment (Fig. 3).
ANF mRNA levels
In the DOCA-salt experiment (Figs. 4-6), ANF mRNA levels in DOCA only, salt only, and rats treated with DOCA-salt had a tendency to be higher than those of the control rats. However, those differences were not statistically significant. In the aortic-banding regression experiment, ANF mRNA levels showed similar results among all groups.
This work shows that ANF gene expression in aortic tissue, as measured by steady-state mRNA levels in two models of hypertension, remained unchanged despite significant anatomical and biochemical hypertrophy, as evidenced by increases in aortic-weight/body-weight ratio and in α2 Na+K+-ATPase expression, respectively. This is in contrast to recent findings made with these models showing tissue-specific regulation of renal and cardiac ANF mRNA levels (12), as demonstrated by increased renal ANF mRNA levels in hypertension induced by aortic banding and a reduction in transcript in DOCA-salt hypertension. In addition, this investigation demonstrated model-specific changes in angiotensinogen mRNA levels consisting of a significant increase of such levels in DOCA-salt rats and a decrease in aortic-banded rats. Decrease of α2 Na+K+-ATPase mRNA levels, a biochemical marker of hypertrophy, was found in animals exhibiting the highest blood pressure levels.
The 31-mm Hg blood pressure differences between control and DOCA-salt rats is comparable to the blood pressure change between sham-operated and aortic-banded rats. In the animals treated with DOCA or salt alone and in the aortic-banded animals treated with low-dose ramipril, blood pressure and aortic-weight/body-weight ratio did not appear to correlate. These findings indicate that anatomical aortic hypertrophy is not always correlated with the hemodynamic load and emphasize the need to use multiple measures of hypertrophic processes.
Herrera et al. (10) reported that 2 and 8 weeks of DOCA-salt treatment increased α1 Na+K+-ATPase mRNA levels and decreased α2 Na+K+-ATPase mRNA levels in the rat aorta. The same group also reported the deinduction of the α2 isoform mRNA levels in the AII-infused and aortic-banded hypertensive rat (11). In our studies, however, DOCA-salt did not induce a change in α1 or α2 Na+K+-ATPase mRNA levels. The reason for this apparent discrepancy may be the differences in treatment and the severity of the hypertension. The former work used a treatment schedule consisting of daily 15 mg/kg DOCA administration, leading to a systolic blood pressure of ≤220 mm Hg in 2 weeks and 170 mm Hg in 8 weeks, whereas in our work, the systolic blood pressure did not reach these values. Transgenic rats expressing a reporter gene driven by the α2 Na+K+-ATPase human gene promoter showed deinduction of both the cardiac and aortic reporter activity in a manner that correlated with systolic pressure >150 mm Hg (11), a value that is higher than the 144 mm Hg obtained in our DOCA-salt rats. In the aortic-banding experiments, however, we found decreased α2 Na+K+-ATPase mRNA levels in the banded rats' carotid arterial blood pressures >150 mm Hg. In the aortic-banding regression experiment, α2 Na+K+-ATPase mRNA levels of the banded rats treated with low-dose ramipril, a treatment that affects local RAS only and is known to reverse cardiac hypertrophy in hypertensive animals (16), also was decreased, even though the aortic-weight/body-weight ratio was normalized. These results suggest that α2 Na+K+-ATPase mRNA levels reflect hemodynamic load rather than anatomic hypertrophy of the aorta. Neither AII nor mineralocorticoid affects α2 Na+K+-ATPase mRNA levels in vascular smooth-muscle cells (17,18).
Aortic angiotensinogen mRNA levels changed in an opposite fashion between the two hypertensive models used in this work. That is, treatment with DOCA, salt, or DOCA-salt increased aortic angiotensinogen mRNA levels, whereas aortic banding decreased these levels. We previously found (8; T. Ogawa, unpublished observations), in the same groups of animals, that treatment with DOCA alone, salt alone, or DOCA-salt significantly depressed plasma renin activity (PRA) and plasma AI and AII levels, whereas in aortic-banded rats, PRA increased slightly, and plasma AII increased significantly (19). Inhibition of both local and circulating RAS by high-dose ramipril in the banded rats resulted in suppressed plasma AII levels and increased PRA. Inhibition of the local RAS by low-dose ramipril did not affect plasma AII or PRA as compared with control or sham-operated animals. These results, together with those presented here, show that aortic angiotensinogen mRNA levels change in an manner opposite to the changes in plasma AII levels. It is not evident what may upregulate angiotensinogen gene expression in the different groups of our DOCA-salt experiments. Klett et al. (20) reported that AII increased angiotensinogen mRNA levels and angiotensinogen secretion in cultured hepatocytes and suggested that AII has a positive-feedback effect on angiotensinogen synthesis. However, in our study, the positive-feedback mechanism by AII does not seem to be a factor in upregulating aortic angiotensinogen gene expression, given the RAS-inhibiting effects of the DOCA-salt treatment.
Aortic angiotensinogen mRNA levels decreased after aortic banding. This decrease was most clearly observed in the prevention experiment. In both cases, treatment with high-dose ramipril, which inhibits both tissue and circulating RAS, resulted in a significant increase in angiotensinogen expression, whereas low-dose ramipril, which inhibits only tissue RAS, failed to reverse the downregulation of aortic angiotensinogen gene expression induced by aortic banding. This was in contrast to previous investigation showing that aortic ACE increases in parallel with the increase in aortic-weight/body-weight ratio (21). This increase was reversed by low-dose ramipril administration, showing that this is a tissue RAS-dependent event and that in our experiments, plasma AII may be more important than the local RAS in suppressing aortic angiotensinogen gene expression. Nevertheless, the upregulation of angiotensinogen gene expression in DOCA-salt rats and its downregulation in aortic-banded animals may be related to a suppression of the local and plasma RAS in the former model and an activation of the local and plasma RAS in the latter. Indeed, in the same experimental models, we found (unpublished observations) that renal ACE gene expression (too low to assess by Northern blot in the aortic tissue samples in this work) follows the opposite pattern described here for aortic angiotensinogen. ACE inhibition or AT-1 blockade in the DOCA-salt model, however, were shown to suppress mesangial expansion and cell proliferation and expression of growth factors, showing that at least in some tissues, local RAS inhibition can have significant effects in this low-renin model (22). Enhanced expression of growth factors and high circulating levels of endothelin (23) may be responsible for the hypertrophic effect of the DOCA-salt treatment in aortic tissue observed in our investigations. The reversal of the increase of the aortic-weight/body-weight ratio by high-dose ramipril suggests that aortic hypertrophy is at least partly due to the proliferative effect of AII on vascular smooth-muscle cells (24).
The ANF mRNA levels were measured by QC-RT-PCR. This newly developed technique (12) is able reliably to quantify ANF mRNA in aorta. The concentration of the ANF mRNA levels in aorta from control rats was found to be 105 times lower than the concentration of ANF mRNA levels in the atrium. However, by radioimmunoassay, ANF prohormone is one third of that in the atrium (7). The discrepancy between ratios of ANF1-126 to ANF mRNA concentration in aorta and atria require further inquiry.
The unchanging levels of aortic ANF mRNA levels in hypertensive rats is in sharp contrast with our previous findings in these animals for cardiac and renal ANF mRNA levels, plasma ANF levels, as well as with a report on changes in aortic ANF-A receptor mRNA levels in experimental hypertension (25). It is possible, however, that, given the observed tendency in the experimental groups of the DOCA-salt experiment to have higher ANF mRNA levels than controls, a more aggressive treatment would have resulted in a significant increase in aortic ANF mRNA levels and a downregulation of α2 Na+K+-ATPase, as observed in animals with >150 mm Hg systolic blood pressure (10). If indeed attained, a significant increase in aortic ANF mRNA after DOCA-salt treatment, together with the fact that aortic banding did not increase aortic ANF mRNA concentration, would indicate that aortic ANF gene expression is regulated in these models in a similar manner as atrial ANF. Atrial ANF gene expression is stimulated by DOCA-salt treatment but appears unaffected by aortic banding (8,9). In contrast, renal ANF mRNA is increased by aortic-banding hypertension and decreased by DOCA-salt treatment (T. Ogawa, unpublished observations).
By using an endothelial cell/smooth-muscle cell coculture system, Kamatsu et al. (26) reported that C-type natriuretic peptide (CNP), which is secreted constitutively from endothelial cells, affects growth inhibition and relaxation of smooth muscle through the guanylyl cyclase-coupled type B natriuretic peptide receptor (GCB receptor). The same group also demonstrated (27) that on switching from a contractile phenotype to a synthetic phenotype, smooth-muscle cells upregulate the GC-B receptor. ANF and BNP circulate in significantly increased amounts in the two types of hypertension studied here (8,28), and both peptides have been reported to stimulate CNP synthesis and release from bovine aortic endothelial cells (29). Therefore it is conceivable that any antihypertrophic effect of ANF at the stages of hypertension studied here may be achieved at least in part by ANF- or B-type natriuretic peptide (BNP)-mediated stimulation of CNP production. In addition, GC-A receptor upregulation was described in the aorta of spontaneously hypertensive rats (SHRs) and in DOCA-salt hypertensive animals (30), so that an additional antihypertrophic effect of increased ANF and BNP plasma levels may be directly mediated by interaction with the increased GC-A receptor expression. Evidence for complementary roles of natriuretic peptides in regulating smooth-muscle cell proliferation is suggested by findings showing differential sensitivities of cells from different vascular origins to ANF, BNP, or CNP (4).
Altogether, the findings discussed suggest a paradigm whereby the natriuretic peptide system provides a modulator role for cardiovascular hypertrophy by either endocrine or paracrine interactions. At the particular hypertensive stages in which our investigations were carried out, regulation of aortic ANF mRNA levels appears largely independent of pressure load, volume load, and plasma and local RAS.
Acknowledgment: We thank Michelle Stevenson, Amalia Ponce, and Carole Frost for their excellent assistance. This work was supported by grants from The Ontario Heart and Stroke Foundation and The Medical Research Council of Canada.
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