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(Hypertension. 1997;30:796-802.)
© 1997 American Heart Association, Inc.

Articles

Molecular Mechanism of Angiotensin II Type I and Type II Receptors in Cardiac Hypertrophy of Spontaneously Hypertensive Rats

Naoki Makino; Masahiro Sugano; Shoji Otsuka; ; Tomoji Hata

From the Department of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan.

Correspondence to Naoki Makino, MD, Department of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, 4546, Tsurumihara, Beppu 874, Japan.

	Abstract

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Abstract We administered angiotensin (Ang) II receptor type 1 (AT₁) blockade (losartan; 10 or 40 mg/kg per day), type II receptor (AT₂) blockades (PD123319; 100 mg/kg per day), or angiotensin-converting enzyme (ACE) inhibitor (enalapril; 30 mg/kg per day) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. At the end of the treatment, high doses of losartan and enalapril significantly reduced the arterial systolic blood pressure compared with the untreated SHR to the level of WKY rats. But low doses of losartan and PD123319 were without effect. High doses of losartan and enalapril also significantly reduced both the left ventricular (LV) weight and the ratio of LV to body weight compared with the untreated SHR, which were still larger than that of WKY rats. However, the collagen concentration of SHR treated with high doses of losartan or enalapril was completely reduced to the level of WKY rats. Using reverse transcription polymerase chain reaction, we examined the mRNA expression for ACE, AT₁, and AT₂ in experimental animals. The enhanced AT₁ mRNA expression was significantly decreased in the SHR treated with a high dose of losartan or PD123319 compared with the untreated SHR. The level of ACE mRNA was also decreased in the SHR treated with a high dose of losartan or enalapril. The level of AT₂ mRNA was not significantly different between the Wistar-Kyoto rats and the SHR; however, this expression was decreased significantly after the treatment with a high dose of losartan or PD123319. These results indicate that AT₁ receptor and ACE, but not AT₂ receptor, play a crucial role in the remodeling of matrix tissue but a smaller role in the development of the hypertrophy of LV myocyte in SHR and that the LV/body weight changes do not fully account for the complete suppression of hypertension.

Key Words: angiotensin II • angiotensin receptor • cardiac hypertrophy • rats, inbred SHR • renin-angiotensin system

	Introduction

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In SHR, both left ventricular hypertrophy and characteristic vascular resistance properties are already present early in life as based on the results of a comparison with normotensive WKY rats, thus suggesting that they play a role in the pathogenesis of hypertension.^{1 2} ACE inhibitors can lower the blood pressure in SHR by reducing Ang II production and decreasing bradykinin degradation,^{3 4} but it is thought that most of the observed actions, affecting both blood pressure and cardiovascular hypertrophy, are caused by Ang II generation.^{5 6} Ang II is, therefore, considered to act as a growth-promoting factor directly on cardiac myocytes, whereas an ACE inhibitor induces the regression of hypertrophied hearts both in experimental and humans either through blood pressure–dependent or –independent mechanisms.^{7 8 9} In fact, ramipril treatment in the renovascular hypertensive rat model was associated with a complete inhibition of cardiac fibrosis. This antifibrotic effect was also present in low doses of ramipril that did not lower blood pressure.¹⁰ Thus, there are several studies in which the effects of the ACE inhibitors on cardiac and vascular functions were independent of their antihypertensive and antihypertrophic actions because they were also observed after subantihypertensive doses of the ACE inhibitors.^{11 12} In addition, the role of Ang II in cardiovascular development is thought to be relatively nonspecific and to play a pressure-independent role that acts directly on the myocardium and vascular smooth muscle.^{13 14 15} These findings thus suggest that the RAS plays an important role in cardiac and vascular development and the process of cardiac hypertrophy in WKY as well as SHR.

Recently, a specific Ang II receptor antagonist has been developed, including those blocking the AT₁ and AT₂ receptor subtypes, with the aim of affecting the RAS more specifically.^{16 17} The antihypertensive action of losartan is based on the blockade of AT₁ receptors, which are believed to mediate most of the cardiovascular actions of Ang II.¹⁸ AT₂ receptors are also thought to have some role in the maintenance of systemic blood pressure and responsiveness of the cardiovascular system to Ang II.^{19 20} However, there is little information regarding the interaction among mRNAs for AT₁ and AT₂ receptors and ACE to the extracellular matrix and the cardiac AT₁ myocyte. The expression of AT₁ and AT₂ receptor mRNAs was further increased in the stretch-induced cardiac myocytes after pretreatment with AT₁ receptor antagonist.²¹

In the present study, we examined the long-term effects of AT₁ antagonist losartan, AT₂ antagonist PD12377, or ACE inhibitor enalapril to selectively block the RAS on the development of cardiac hypertrophy in SHR. We then measured not only the LV/BW but also collagen concentration in the heart and the regulation of the molecular expression of cardiac mRNA levels for AT₁ receptor, AT₂ receptor, and ACE. These provide the information on how the RAS affects extracellular matrix and cardiac myocytes, as well as some clues about the cardiac RAS in LV hypertrophy. We also administered losartan at subantihypertensive doses and a low dose of losartan to SHR in the study.

	Methods

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Animals and Experimental Protocols
Male SHR at 5 weeks of age (n=60) and age-matched male WKY (each group, n=12) as genetically normotensive control strains were used for this study. All animals were housed in a room where temperature, humidity, and light were controlled, and a standard rat diet plus water was provided ad libitum. The systolic blood pressure and heart rate were measured once a week using the tail-cuff method, and the body weight was also checked. The AT₁ receptor antagonist losartan or the AT₂ receptor antagonist PD123319²² was administered daily to SHR from 10 weeks of age, when cardiac hypertrophy had not yet developed, and the addition of the agent was continued until 20 weeks of age. The concentration of losartan was administered at either low (10 mg/kg per day, n=12) or high (40 mg/kg per day, n=12) doses, whereas PD123319 was given at 100 mg/kg per day (n=12). Other SHR were treated with the ACE inhibitor enalapril (30 mg/kg per day, n=12) or vehicle (n=12) from 10 weeks of age. All drugs were administered daily to SHR through a stomach tube. At the end of the treatment with each drug, the body weight and the blood pressure were measured. Next, the rats were killed by decapitation, and blood samples were drawn into chilled tubes (4°C) containing 0.1% EDTA for the plasma ACE activity. The left ventricle was immediately removed, washed with ice-cold 10 mmol/L potassium phosphate buffer (pH 8.3), weighed, and prepared to determine the tissue ACE activity, collagen contents, Ang II receptor assays, and RNA isolation. The hearts were then frozen in liquid nitrogen and kept for up to 2 weeks at -80°C until the assays were performed.

Measurement of ACE Activity
The ACE activity was measured by the modified method of Hayakari et al.²³ Briefly, all tissue samples were diced and homogenized in the same ice-cold buffer. The homogenate was centrifuged (5000g) for 10 minutes, and the supernatant was dialyzed for 24 hours against 20 vol of the same buffer at 4°C. The dialyzed supernatant fluid was then used for the tissue ACE activity. For the plasma ACE activity, a 1:5 dilution of the plasma was used. The assay for the ACE activity was carried out in a 150-mL incubation mixture containing 80 mmol/L potassium phosphate buffer (pH 8.3), 0.6 mol/L sodium chloride, 3 mmol/L hypuryl-histidyl-L-leucine, and the sample. The reaction was initiated by the addition of the substrates at 37°C for 30 minutes. The reaction was terminated by immersion of the test tubes into a boiling-water bath for 10 minutes. The enzyme activity in the resulting supernatant fluid was determined from the absorbance at 382 nm by the differential spectrophotometric method. The control run was identical to the above procedure minus the incubation.

Measurement of Collagen Concentration
The myocardial collagen content was measured by the hydroxyproline concentration of tissue, as described by Bergman and Loxley.²⁴ After the heart was dried for 24 hours, the specimens were hydrolyzed in 6N hydrochloric acid solution at 100°C. Then p-dimethylamino-benzaldehyde (Ehrlich's reagent) dissolved in buffer at pH 7.0 was added to form a complex with hydroxyproline. The concentration of hydroxyproline was measured by a spectrophotometric analysis at a wave length of 558 nm. The collagen content was estimated by multiplying the hydroxyproline content by a factor of 8.2.²⁵ The concentration of collagen was expressed as milligrams of collagen per gram of wet weight.

Quantitative Reverse Transcription Polymerase Chain Reaction Assay
Total RNA was isolated from homogenized LV tissue specimens by a modification of the acid guanidine thiocyanate technique (RNazol, Cinna/Biotecx). After RNA was pelleted by ultracentrifugation, it was resuspended in 0.2 mol/L sodium acetate at pH 5.5 and shaken at 4°C for 1 hour and then precipitated in 2 vol ethanol. The precipitated RNA was dissolved in water, and the amount was quantified by absorbance at 260 nm in duplicate measurements. The relative mRNA levels were then compared with the total amount of the applied RNA. The RT-PCR method was used to detect mRNA for an ACE and Ang II receptor in the LV tissue of the experimental rats as described previously²⁶ by us. Briefly, 1 µg of total RNA was reverse transcribed into cDNA and then amplified using an RT-PCR kit (Life Technologies, Inc). A nonradiolabeled system (Life Technologies, Inc) was used to label the PCR product with biotinylated 14-dCTP. The amplification profile involved denaturation at 95°C for 1 minute, annealing at 64°C for 1 minute, and extension at 72°C for 1 minute. The PCR product from each primer set consists of different oC base pairs (bp), depending on the primers used. The PCR products are designed not to overlap in size. The ranges of the cycles that were exponential in each mRNA and the PCR cycles for quantitating each mRNA were then determined. The sense and antisense primers were synthesized by model 352 and 394 DNA/RNA synthesizers (Applied Biosystems). For the AT₁ receptor,²⁷ the sense primer was 5'-GCCAAAGTCACCTGCATCAT-3', and the antisense primer was 5'-AATTTTTTCCCCAGAAAGCC-3'. The PCR product size was 494 bp, and there were 26 PCR cycles in mRNA. For the AT₂ receptor,²⁸ the data were: sense, 5'-TGAGTCCGCATTTAACTGC-3'; antisense, 5'-ACCACTGAGCATATTTCTCGGG-3'; and size, 476 bp in 28 cycles. For ACE,²⁶ the data were: sense, 5'-GACTGGTCCAACATCTATG-3'; antisense, 5'-ATGAAGCTGACGAAGTACCT-3'; and size, 739 bp in 28 cycles. As an internal control for input RNA, a PCR oligonucleotide primer for GAPDH was chosen at 349 bp from the rat²⁹ : sense, 5'-CATGGTCTACATGTTCCAGT-3' and antisense, 5'-GGCTAAGCAGTTGGTGGTGC-3' in 12 cycles. After the completion of PCR, 2 mL of each PCR product from the same animal was applied to the same well of a 12.5% polyacrylamide gel (Daiichi-Kagaku Pharmaceuticals). Electrophoresis was performed at a constant 20 mA for 90 minutes in 25 mmol/L Tris and 0.2 mol/L glycine. After electrophoresis, the PCR products were electrophoretically transferred to a nylon membrane (Stratagene) in 0.2x TBE at 0.4 mA/cm² for 90 minutes. A semidry electroblotting apparatus (Advantec) was used. The blots were analyzed and visualized with a biotin detection kit (Millipore). The density of each PCR band was analyzed with a densitometer (model 620, Japan Bio-Rad). The amount of mRNA was described as the ratio to GAPDH.

Ang II Receptor Assay
The ventricular membranes from the rat hearts were prepared using a modification of the method of Baker et al.³⁰ A tissue homogenate was prepared with 0.25 mol/L sucrose and 25 mmol/L Tris (pH 7.5) containing 0.5 mmol/L EDTA, 0.5 mmol/L PMSF, 10 mg/L bacitracin, 4 mg/mL leupeptin, 4 mg/mL pepstatin, and 40 U/mL trasylol. A Polytron homogenizer was used twice for 30 seconds each at half-maximal speed. The homogenate was sedimented twice at 10 000g for 20 minutes, and the supernatant was then centrifuged at 45 000g for 30 minutes. The pellet was resuspended in 0.6 mol/L KCl and 30 mmol/L histidine (pH 7.0), again containing 0.5 mmol/L EDTA, 0.5 mmol/L PMSF, 10 mg/L bacitracin, 4 mg/mL leupeptin, 4 mg/mL pepstatin, and 40 U/mL trasylol. This was then resedimented at 45 000g for 30 minutes. The pellets obtained from the final centrifugation were washed three times and resuspended in 25 mmol/L Tris (pH 7.5) containing 10 mmol/L MgCl₂, 0.5 mmol/L PMSF, 10 mg/L bacitracin, 4 mg/mL leupeptin, 4 mg/mL pepstatin, and 40 U/mL trasylol; a hand-driven glass/glass homogenizer was used. All centrifugations were performed at 4°C. The membrane preparations were immediately frozen in liquid nitrogen and held in aliquots at -80°C until use. The yield was approximately 1 mg of protein per gram of heart. Membrane protein (120 to 140 µg) was incubated with increasing concentrations of the radiolabeled Ang II antagonist ¹²⁵I-[Sar¹,Ile⁸]Ang II (specific activity, 2200 Ci/mmol; New England Nuclear). The incubation mixture was composed of the assay buffer containing ¹²⁵I-[Sar¹,Ile⁸]Ang II and a competing unlabeled Ang II antagonist. The assay buffer was 25 mmol/L Tris (pH 7.5) containing 10 mmol/L MgCl₂, 2 g/L bovine serum albumin, 10 mg/L bacitracin, 0.5 mmol/L PMSF, and 1 mg/mL of each peptidase inhibitor antipain, phosphoramidon, leupeptin, pepstatin, and amastatin.³¹ The reaction was initiated by the addition of the aliquot of membrane protein and then continued for 60 minutes at 37°C. Saturation isotherms had increasing concentrations of ¹²⁵I-[Sar¹,Ile⁸]Ang II between 0.1 and 2 nmol/L. Binding assays were done in duplicate or triplicate. Specific binding was defined as the portion of total counts displayed by 1 µmol/L [Sar¹,Ile⁸]Ang II. At ligand concentrations equivalent to the affinity of the receptor for the radioligand, specific binding averaged 85%.

Reagents and Statistical Method
All reagents were purchased from Sigma Chemical Co, unless otherwise indicated below. Losartan was a gift of Merck, Sharp & Dohme Research Laboratories. PD123319 was kindly provided by Parke-Davis Pharmaceutical Research Division. All data are given as the mean±SEM, and the data were evaluated using ANOVA and unpaired Student's t test as appropriate. Statistical significance was accepted at P<.05.

	Results

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Table 1 summarizes the characteristics of the WKY and SHR at the end of the treatment with either Ang II receptor blockades or ACE inhibitor. In the untreated SHR, both the LV weight and the LV/BW ratio were significantly (P<.05) higher than those of the WKY aged 20 weeks. The systolic blood pressure in the 20-week-old SHR was also higher than that in the WKY. The administration of a high dose of losartan or enalapril to SHR normalized the systolic blood pressure, which was brought to the levels of WKY. However, both the LV weight and LV/BW ratio in the treatment were significantly reduced compared with the untreated SHR but remained well above the level of the WKY. On the other hand, the administration of 10 mg/kg losartan or PD123319 had no effect on the LV weight or the LV/BW ratio. Thus, the low dosage of losartan or AT₂ blockade did not reduce the blood pressure and did not decrease the weight of LV in SHR.

View this table: [in this window] [in a new window]   Table 1. LV Weight, LV to Body Weight Ratio, and Systolic Blood Pressure at 20 Weeks of Age in Experimental Rats

Table 2 shows the ACE activity in the experimental animals from the plasma and the LV tissue at 20 weeks of age. The ACE activity in the untreated SHR was significantly increased by 74% in the plasma and by 50% in the LV tissue when compared with the WKY. These activities were not significantly different between the untreated SHR and the treated SHR with losartan or PD12377. However, the ACE activity in the SHR treated with enalapril was significantly reduced in both plasma (81%) and LV (77%) tissue. The collagen concentration in LV tissue from each group was also studied. The collagen concentration significantly decreased in the samples obtained from SHR treated with a high dose of losartan (18%) or enalapril (20%) compared with the untreated SHR. However, with the low dose of losartan or PD12377, there was no effect.

View this table: [in this window] [in a new window]   Table 2. Plasma and Myocardial ACE Activity and Myocardial Collagen Concentration at End of Treatment for Each Drug in Experimental Rats

The levels of ACE and AT₁ and AT₂ mRNA expression were detected in the LV myocardium of rats in each experimental group by the RT-PCR method. These approaches were used to establish the validity of the quantitative aspect of RT-PCR. Using this approach, it was thus possible to document differences in the level of each mRNA in the LV tissue. The GAPDH signals used as an internal control were unchanged in these experimental rats. An apparently higher level of AT₁ mRNA was also seen in the myocardium of the SHR (Fig 1), whereas with a high dose of losartan, the mRNA level of the SHR significantly restored the level of the WKY. Although the levels of AT₁ mRNA did not change in the SHR treated with a low dose of losartan or enalapril, this gene expression was significantly reduced with PD123319 compared with the untreated SHR. The results of the AT₂ mRNA expression are shown in Fig 2. The AT₂ mRNA was also low and not significantly different from WKY and untreated SHR. However, the administration of losartan or PD123319 to the SHR reduced the level of AT₂ mRNA compared with the untreated SHR. The ACE mRNA increased in the untreated SHR compared with the WKY (Fig 3). The level of ACE mRNA also significantly decreased in the SHR after being treated with a high dose of losartan or enalapril. The ACE mRNA expression by PD12377 treatment was also more enhanced than that in the untreated SHR. The results of the Ang II receptor binding assays are shown in Fig 4.

View larger version (57K): [in this window] [in a new window]   Figure 1. Top, Detection of AT1 mRNA by RT-PCR in the LV from WKY, SHR, and SHR treated with each drug from 10 to 20 weeks. GAPDH mRNA is an internal control. MM indicates molecular weight marker. Bottom, Results regarding the amount of mRNA after analysis of its ratio to GAPDH. Each bar represents the mean±SEM of six experiments. Los (high) and Los (low) indicate the SHR group treated with a high concentration of losartan (40 mg/kg per day) and a low concentration of losartan (10 mg/kg per day), respectively. PD indicates PD123319; EP, enalapril. *P<.05 vs WKY; P<.05 vs untreated SHR.

View larger version (61K): [in this window] [in a new window]   Figure 2. Top, Detection of AT2 mRNA by RT-PCR in the LV from WKY, SHR, and SHR treated with each drug. GAPDH mRNA is an internal control. MM indicates molecular weight marker. Bottom, Results of the amount of mRNA after analysis of its ratio to GAPDH. Each bar represents the mean±SEM of six experiments. Los indicates losartan; PD, PD123319; and EP, enalapril. *P<.05 vs WKY; P<.05 vs untreated SHR.

View larger version (57K): [in this window] [in a new window]   Figure 3. Top, Detection of ACE mRNA by RT-PCR in the LV from WKY, SHR, and SHR treated with each drug. GAPDH mRNA is an internal control. MM indicates molecular weight marker. Bottom, Results of the amount of mRNA after analysis of its ratio to GAPDH. Each bar represents the mean±SEM of six experiments. Los indicates losartan; PD, PD123319; and EP, enalapril. *P<.05 vs WKY; P<.05 vs untreated SHR.

View larger version (18K): [in this window] [in a new window]   Figure 4. Bar graphs show the Ang II receptor densities in the membranes isolated from the heart of WKY or SHR treated with or without each drug. Each value is the mean±SEM of six experiments. Los (high) and Los (low) indicate the SHR group treated with a high concentration of losartan (40 mg/kg per day) and a low concentration of losartan (10 mg/kg per day), respectively. PD indicates PD123319; EP, enalapril. *P<.05 vs WKY; P<.05 vs untreated SHR.

The maximum number of Ang II receptors significantly increased in the myocardium of the untreated SHR compared with the same number in the WKY. Although the maximum number of Ang II receptors was significantly downregulated in the membranes from the SHR with a high dose of losartan compared with those of the untreated SHR group, these values were not significantly different among the membranes of the SHR treated with a low dose of losartan, PD12377, or enalapril. The dissociation constant in these assays also did not differ among any experimental groups.

	Discussion

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The present study demonstrates that chronic treatment with a high dose of losartan and enalapril normalized the systolic pressure whereas the other treatments were without effect and that LV/BW, which remained well above the level of the WKY, was only attenuated by the high dose of losartan and enalapril. In contrast, the collagen concentration in the heart was nearly normalized by the high dose of losartan and enalapril, suggesting that RAS has more important actions on extracellular matrix than the cardiac myocyte size, ie, the antihypertrophic action produced by the AT₁ antagonist. We can speculate that this may be important for both the blood pressure reduction or the reversal of LV hypertrophy in SHR. This effect of a high dose of losartan was similar to that observed with the ACE inhibitor.^{32 33} The hypertrophy in untreated SHR, in which LV/BW was increased at 20 weeks, was associated with increased amounts of ACE mRNA, as described previously in ventricular hypertrophy by Schunkert et al.³⁴ This suggests that ACE is directly involved in the hypertrophy. All the treated SHR in the present study were given drugs between the ages of 10 to 20 weeks. By 10 weeks, LV hypertrophy and changes in the vascular media are already established; thus, the treatment was at least partly aimed at reversing established pathology, much as in human patients with hypertension. Our study suggests that the reversal of myocyte hypertrophy with the current protocol was only partial and that reversal of the excess amounts of collagen and matrix tissue is an important action of losartan and ACE inhibitors. Thus, RAS appears to play an important role in the production of these constituents in the heart of SHR. Our findings differ from others,¹³ where the treatment began at 4 weeks and the cardiovascular system was immature. As shown in Fig 1, an apparent increase in AT₁ mRNA levels was seen in 20-week-old SHR, whereas AT₂ mRNA levels in these hypertensive rats appeared unchanged. The administration of a high dose of losartan to SHR normalized the increased AT₁ mRNA levels. These observations were similar to the results described previously by Suzuki et al.⁶ However, in the present study, a high dose of losartan downregulated both AT₂ and ACE mRNAs in SHR. These findings may imply that losartan inhibited Ang II binding not only to AT₁ receptor but also to AT₂ receptor in the regulation of hypertrophy in the in vivo model or that there could be some interactions between their signaling pathways. On the other hand, in vitro stretching myocytes upregulated both AT₁ and AT₂ receptor mRNA levels, and this effect was completely blocked by AT₁ receptor antagonist but not AT₂ receptor antagonist.³⁵ These discrepancies may contribute to the differences among experimental models used in the studies. The normalization of LV ACE mRNA by both a high dose of losartan and enalapril is a different pattern from that of AT₁ receptor mRNA, including AT₁ receptor mRNA, which differed little from those of untreated SHR. Presumably, ACE mRNA reflects the local (cardiac) RAS to a greater degree than AT₁ receptor mRNA.

Recent studies have shown that treatment with both ACE inhibitors and AT₁ receptor antagonists was associated with a regression of cardiac hypertrophy and fibrosis in animal models of SHR and renovascular hypertension as well as hypertensive patients.^{6 18 36} Others have shown that the administration of AT₁ receptor antagonists is associated with regression of vascular hypertrophy and reduction of media/lumina ratio of the vessels.³⁷ AT₁ blockade induced cardiac regression and a fall in blood pressure in SHR, in contrast to hydralazine, despite the greater blood pressure reduction with the latter drug.³⁸ These observations thus suggest the regression of cardiac hypertrophy and inhibition of ventricular remodeling to be mainly carried out by the inhibition of the effects of Ang II in cardiac tissue. The present study also showed that enalapril treatment also prevented the development of cardiac hypertrophy and reduced blood pressure, although we did not examine the effects of enalapril at subantihypertensive doses. The results obtained with enalapril were similar to those obtained for losartan observed here. The regression of both cardiac hypertrophy and fibrosis was found in SHR rats treated with an ACE inhibitor, quinapril, for 20 weeks.³⁹ In a renovascular hypertension rat model, ramipril treatment at subantihypertensive doses was associated with a complete inhibition of cardiac fibrosis,¹⁰ which thus suggests that the signaling mechanisms for cardiac fibrosis may be independent of the blood pressure.

These results also demonstrate that the RAS plays an important role in both the development of LV hypertrophy and in the progression of ventricular remodeling. PD123319 did not affect either the systolic blood pressure or the LV weight and collagen concentration in the present study. These results thus indicate that the AT₂ receptor does not play an important role in cardiac hypertrophy. It is thus suggested that Ang II action in the heart is mediated through the AT₁ receptor rather than the AT₂ receptor and therefore may be more important in cardiac growth and the development of hypertrophy. The physiological functions of AT₂ receptors remain unclear^{40 41} ; this receptor is present only at low levels in many adult tissues, including the rat heart,⁴² although it is abundantly expressed in many fetal tissues.⁴³ It was reported recently that the AT₂ receptor modulates the antigrowth effects on vascular smooth muscle,⁴⁴ endothelial cells,⁴⁵ and programmed cell death.⁴⁶ The present study showed that AT₂ mRNA was similar in 20-week-old SHR and WKY. However, in the present study, AT₁ blockade significantly reduced the expression of AT₁ and AT₂ receptors. Therefore, the role of AT₂ receptors in cardiac hypertrophy remains unclear.

In conclusion, the present study indicates that ACE and AT₁ receptors but not AT₂ receptors play a crucial role in the development of LV hypertrophy in SHR, which affects not only the myocytes but also the production of collagen and other matrix tissues. If similar actions take place in human essential hypertension, it may provide this class of drugs with an edge over others used in the treatment of hypertension.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme Ang = angiotensin AT1, AT2 = angiotensin II type 1, type 2 (receptor) LV/BW = left ventricle/body weight ratio RAS = renin-angiotensin system RT-PCR = reverse transcription polymerase chain reaction SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan. We thank S. Taguchi for valuable technical assistance.

Received February 25, 1997; first decision March 12, 1997; accepted March 25, 1997.

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